Browsing by Person "Seifert, Jana"

Now showing 1 - 16 of 16

The active core microbiota of two high-yielding laying hen breeds fed with different levels of calcium and phosphorus
(2022) Roth, Christoph; Sims, Tanja; Rodehutscord, Markus; Seifert, Jana; Camarinha-Silva, Amélia
The nutrient availability and supplementation of dietary phosphorus (P) and calcium (Ca) in avian feed, especially in laying hens, plays a vital role in phytase degradation and mineral utilization during the laying phase. The required concentration of P and Ca peaks during the laying phase, and the direct interaction between Ca and P concentration shrinks the availability of both supplements in the feed. Our goal was to characterize the active microbiota of the entire gastrointestinal tract (GIT) (crop, gizzard, duodenum, ileum, caeca), including digesta- and mucosa-associated communities of two contrasting high-yielding breeds of laying hens (Lohmann Brown Classic, LB; Lohmann LSL-Classic, LSL) under different P and Ca supplementation levels. Statistical significances were observed for breed, GIT section, Ca, and the interaction of GIT section x breed, P x Ca, Ca x breed and P x Ca x breed (p < 0.05). A core microbiota of five species was detected in more than 97% of all samples. They were represented by an uncl. Lactobacillus (average relative abundance (av. abu.) 12.1%), Lactobacillus helveticus (av. abu. 10.8%), Megamonas funiformis (av. abu. 6.8%), Ligilactobacillus salivarius (av. abu. 4.5%), and an uncl. Fusicatenibacter (av. abu. 1.1%). Our findings indicated that Ca and P supplementation levels 20% below the recommendation have a minor effect on the microbiota compared to the strong impact of the bird’s genetic background. Moreover, a core active microbiota across the GIT of two high-yielding laying hen breeds was revealed for the first time.
Adaptations of Prevotella bryantii B14 to short-chain fatty acids and monensin exposure
(2023) Trautmann, Andrej; Seifert, Jana
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.
Central carbon metabolism, sodium-motive electron ransfer, and ammonium formation by the vaginal pathogen Prevotella bivia
(2021) Schleicher, Lena; Herdan, Sebastian; Fritz, Günter; Trautmann, Andrej; Seifert, Jana; Steuber, Julia
Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na+-translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na+-dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus. In P. bivia, the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis, another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.
Dynamic development of viral and bacterial diversity during grass silage preservation
(2023) Sáenz, Johan S.; Rios-Galicia, Bibiana; Rehkugler, Bianca; Seifert, Jana
Ensilaging is one of the most common feed preservation processes using lactic acid bacteria to stabilize feed and save feed quality. The silage bacterial community is well known but the role of the virome and its relationship with the bacterial community is scarce. In the present study, metagenomics and amplicon sequencing were used to describe the composition of the bacterial and viral community during a 40-day grass silage preservation. During the first two days, we observed a rapid decrease in the pH and a shift in the bacterial and viral composition. The diversity of the dominant virus operational taxonomic units (vOTUs) decreased throughout the preservation. The changes in the bacterial community resembled the predicted putative host of the recovered vOTUs during each sampling time. Only 10% of the total recovered vOTUs clustered with a reference genome. Different antiviral defense mechanisms were found across the recovered metagenome-assembled genomes (MAGs); however, only a history of bacteriophage infection with Lentilactobacillus and Levilactobacillus was observed. In addition, vOTUs harbored potential auxiliary metabolic genes related to carbohydrate metabolism, organic nitrogen, stress tolerance, and transport. Our data suggest that vOTUs are enriched during grass silage preservation, and they could have a role in the establishment of the bacterial community.
Effects of diets with different phosporus availability on the intestinal microbiota of chickens and pigs
(2019) Tilocca, Bruno; Seifert, Jana
In the research works of the present thesis, 16S rRNA gene sequencing and metaproteomics were employed to investigate the gut microbiota of chickens and pigs kept at experimental diets with varying amount of calcium-phosphorus (CaP) and supplemented MP. This represents a valuable approach to investigate the bacterial specimens involved in the P absorption, allowing for a comprehensive understanding of how the intestinal bacteria adapt to a new diet and which metabolic routes are affected by changing levels of supplemented P and/or MP. Two major experimental trials were performed during the investigation. The first one was conducted on chickens operating a modulation in the dietary levels of Ca, P and MP. This trial highlighted a shift in the composition of the crop and ceca-associated microbial community depending on the composition of the diet fed. Also, investigated protein inventory revealed that the stress condition due to the reduced P availability is mirrored in the gastrointestinal tract (GIT)-associated microbiota. Marked differences were observed in the functions of the bacterial community in the case of P-available diets versus P-deficient ones. Protein repertoire of the first case draws a thriving microbial community focused on complex and anabolic functions. Contrariwise, the bacterial community in the case of P-lacking diets appears to deal with catabolic functions and stress response. The second trial was conducted on pigs and attempts to define the dynamics featuring the microbiota adaptation to a new challenging diet composed of different protein sources and varying levels of Ca and P. Statistical evidences reveal a stepwise adaptation of the fecal microbiota to the experimental diets fed. Both DNA-based approach and metaproteomics independently reveal three main adaptation phases: -before the feeding of the experimental trial (i.e. Zero), -the response of the microbial community to the challenging factor (i.e. MA) and, finally, - the newly achieved homeostatic balance (i.e. EQ). As observed in the first trial, feeding of the experimental diets impairs the overall fecal microbiota composition, stimulating the presence of phase-specific bacterial specimens and a characteristic relative abundance of the shared ones. Bacterial families responsible for the phase-specific architecture of the fecal microbiota are also active in the biochemical pathways driving the functional peculiarities of each adaptation phase. A deeper investigation of the identified protein repertoire revealed that the observed statistical differences among the adaptation phases are uniquely due to the Ca and P composition of the diets fed. None of the observed effects can be attributed to the diverse protein sources supplemented with the diets. Functional categorization of the identified protein inventory depicts three diverse functional assets of the microbial community. Specifically, prior the feeding of the experimental diets, bacteria are hypothesized to live under homeostatic condition, since they appear to be involved in complex and highly-specialized functions. Following the administration of the experimental diets microbial community changes its functional priority and reduce the expression of highly specialized functions to focus on more essential ones. Proteins involved in complex functions such as widening the substrates array and facing complex sugars tend to increase in abundance while the new homeostatic balance is achieved. Altogether, data from both trials provide useful information for future studies aimed to design effective breeding strategies finalized to reduce the P supplementation in the routinely breeding of livestock and maintain a balanced microbial activity in the animal GIT. Investigation of the dynamics of the porcine microbiota provides instructions on the minimal exposure time required from the intestinal microbiota to adapt to a new dietary composition. This is of fundamental importance for the design of future studies aimed to confirm and/or continue our results. Moreover, the anatomical and physiological similarities occurring between humans and pigs, make our findings of interest for future human nutritional studies, where the mechanisms and lasts of the microbiota adaptation process is still object of discussion.
Evolution of rumen and oral microbiota in calves is influenced by age and time of weaning
(2021) Amin, Nida; Schwarzkopf, Sarah; Kinoshita, Asako; Tröscher-Mußotter, Johanna; Dänicke, Sven; Camarinha-Silva, Amélia; Huber, Korinna; Frahm, Jana; Seifert, Jana
Background: The rumen bacterial communities are changing dynamically throughout the first year of calf’s life including the weaning period as a critical event. Rumen microbiome analysis is often limited to invasive rumen sampling procedures but the oral cavity of ruminants is expected to harbour rumen microbes due to regurgitation activity. The present study used buccal swab samples to define the rumen core microbiome and characterize the shifts in rumen and oral microbial communities occurring as result of calf’s age as well as time of weaning. Results: Buccal swab samples of 59 calves were collected along the first 140 days of life and compared to stomach tubing sample of the rumen at day 140. Animals were randomly divided into two weaning groups. Microbiota of saliva and rumen content was analysed by 16S rRNA gene amplicon sequencing. Our study showed that most rumen-specific bacterial taxa were equally observed in rumen samples as well as in the buccal swabs, though relative abundance varied. The occurrence of rumen-specific OTUs in buccal swab samples increased approximately 1.7 times from day 70 to day 140, indicating the gradual development of rumen as calf aged. The rumen-specific bacterial taxa diversity increased, and inter-animal variations decreased with age. Early weaning (7 weeks of age) rapidly increased the rumen microbial diversity from pre- to post-weaned state. Rumen microbiota of early-weaned calves seemed to have a suppressed growth of starch- and carbohydrate-utilizing bacteria and increased fibre degraders. Whereas, in late-weaned calves (17 weeks of age) no impact of dietary modifications on rumen microbiota composition was observed after weaning. Oral-specific bacterial community composition was significantly affected by calf’s age and time of weaning. Conclusions: The present study showed the significant impact of calf’s age and weaning on the establishment of rumen- and oral-specific bacterial communities utilizing buccal swab samples. The results emphasize the possibility of using buccal swab samples as a replacement of complex stomach tube method for large-scale predictive studies on ruminants. For in-depth rumen microbiome studies, the time of sampling should be carefully considered using an active phase of regurgitation.
Factors influencing proteolysis and protein utilization in the intestine of pigs: A review
(2021) Kurz, Alina; Seifert, Jana
Pigs are among the most important farm animals for meat production worldwide. In order to meet the amino acid requirements of the animals, pigs rely on the regular intake of proteins and amino acids with their feed. Unfortunately, pigs excrete about two thirds of the used protein, and production of pork is currently associated with a high emission of nitrogen compounds resulting in negative impacts on the environment. Thus, improving protein efficiency in pigs is a central aim to decrease the usage of protein carriers in feed and to lower nitrogen emissions. This is necessary as the supply of plant protein sources is limited by the yield and the cultivable acreage for protein plants. Strategies to increase protein efficiency that go beyond the known feeding options have to be investigated considering the characteristics of the individual animals. This requires a deep understanding of the intestinal processes including enzymatic activities, capacities of amino acid transporters and the microbiome. This review provides an overview of these physiological factors and the respective analyses methods.
Impact of age and weaning time on the gut microbiome and the potential host-microbe interactions in calves
(2021) Amin, Nida; Seifert, Jana
The period from birth until the end of weaning is critical for calves as they undergo extreme stress caused by maternal separation, transportation, and weaning related dietary shifts, that can cause long-lasting effects on animal behaviour, health as well as future production parameters. Monitoring the development of microbial ecosystem throughout the gastrointestinal tract of calves and host-microbe interactions during the challenging life periods such as perinatal and weaning is essential for sustainable ruminant production. The present thesis provided new insight on the suitability of buccal swabs as an alternative to complex stomach tubing method for predictive analysis of rumen microbial communities. The changes in oral, rumen and faecal microbial community structure of female German Holstein calves from 8-days to 5-months of age as well as during early- and late-weaning event were identified. The oral microbiota plays a crucial role in animal health. A high dominance of oral pathogens was observed during the first 11-weeks of calves’ life. Similar to the oral microbiota, faeces of 8-day-old calves also showed high abundances of certain opportunistic pathogenic bacteria. Both oral and faecal pathogens showed a decrease in abundance with age and after weaning event in the earlyC group, indicating the age and weaning-dependent maturation of the host immune system. The establishment of dense microbial communities in the faeces of 8-day-old (experimental day 1) pooled herd milk and milk replacer fed Holstein calves was shown and it was dominated by phyla Firmicutes and Actinobacteria and potential lactose- and starch-degrading bacterial species, but as the calves aged and became more mature (5-months of age), their rumen and faecal bacterial communities were dominated by potential fibre-utilizing bacterial genera. The weaning related dietary transitions are critical for calves as their gastrointestinal tract undergoes several modifications, enabling them to digest plant-based diet during the postweaning period. Thus, it was proposed that the age at which animals should be weaned must be carefully considered as it clearly impacted the gastrointestinal tract microbial communities and plasma metabolic profiles of calves in the present study. Early introduction of roughages in the diet of 7-week-old calves increased the abundances of plant fiber degrading bacteria and decreased the abundances of potential lactose- and starch-degrading bacteria in the buccal cavity, rumen and faeces, indicating the weaning-related increase in fiber ingestion and the decrease in milk consumption of the early-weaned group. However, when roughages were introduced in the diet of late-weaned calves at 17-weeks of age, no significant modifications in the structure of gastrointestinal tract microbial communities were observed. Similar to the microbiome, plasma metabolic profiles of early-weaned calves during days 42–112, showed lower concentrations of most of the amino acids, few biogenic amines, and sphingomyelins as compared to the late-weaned calves, suggesting that the liquid diet could provide certain metabolites that can be transported into the bloodstream through gastrointestinal tract. Similarly, the weaning-dependent changes in the quantity of dietary protein, fat and carbohydrates resulted in substantial changes in amino acid metabolism of the early-weaned group. The early-weaning event not only impacted the host microbiome and metabolome but also the host-microbe metabolic interactions as the abundances of potential lactose- and starch degrading bacteria and plasma concentrations of amino acid, biogenic amines and sphingomyelins were strongly positively correlated, both were negatively impacted by the early-weaning event. Thus, it can be concluded that late-weaning was beneficial as it allowed better adaptability of microbes to weaning-related dietary shifts, perhaps due to the greater maturation of their gastrointestinal tract with age as compared to the early-weaning group.
Metaomic studies of the dietary impact on the structural and functional diversity of the rumen microbiome
(2018) Deusch, Simon; Seifert, Jana
Ruminant production efficiency and related emission of greenhouse gases are mainly determined by the rumen microbiome. The structure and activity of the microbial communities in turn are mostly influenced by the animal’s feed intake. The most widely used forage sources for ruminant production in Europe are corn silage, grass silage and grass hay. Progress in animal production requires optimized feeding strategies which presuppose an improved understanding of the dietary impact on the complex bionetwork residing in the rumen. A broad range of different methods are applicable to investigate archaea and bacteria which represent the most active members of the rumen microbiome. Most rumen studies available are restricted to nucleic acid-based approaches with limited functional insights. To improve knowledge about the prokaryotic communities and their adaptation responses to different animal feeds, it is essential to focus on the actual functions out of numerous possibilities that are encoded by the genomes of the rumen microbiome. Therefore proteins are best suited since representing the actual function of investigated cells combined with phylogenetic information. The major aim of this project was the feasible, first-time establishment of a metaproteomics-based characterization of the ruminal prokaryotic communities to further investigate the dietary impact on the prokaryotic rumen metaproteome. The first part was providing an overview about research that used state of the art technologies to investigate the microbiome of the gastrointestinal tract of farm animals. Yet, Omics-technologies and their combination are rarely employed in livestock science. The considered studies relied mainly on stand-alone, DNA-based molecular methods which clearly emphasized the importance of introducing contemporary methods such as shotgun metaproteomics to study the rumen microbiome and to gain deeper, more complete insights into the actual functions carried out by the specific members of the prokaryotic communities. The second part of the current project focused on a suitable, mass spectrometry-based analysis of the prokaryotic communities in the rumen ecosystem. Metaproteomic studies are challenged by the heterogeneity of the rumen sample matter that contains, besides archaeal and bacterial cells, also eukaryotic cells of rumen fungi and protozoa as well as enormous amounts of plant cells from ingested feed and epithelial cells of the animals. Shotgun metaproteomic studies require the extraction of proteins preferably of the desired target organisms to increase the coverage of the respective metaproteome and the reliability of subsequent protein identifications. This entails the avoidance of undesired proteins present in the rumen samples. In contrast to nucleic acids, proteins cannot be enriched or amplified by PCR thus, optimized sample preparation protocols are necessary in order to retrieve enhanced amounts of prokaryotic instead of plant-derived or other eukaryotic cells before protein extraction and subsequent LC-MS/MS analysis. The final step and the major aim of this project was the in depth analysis of the metaproteome of archaea and bacteria and their adaptive response to the most common forages, corn silage, grass silage and grass hay accessing as well host-related influences and variations between different ecological niches within the rumen. Improved mass-spectrometric measurements and the construction of a customized, sample-specific in-house database for enhanced bioinformatic quantification of proteins yielded comprehensive datasets comprising 8,163 bacterial and 358 archaeal proteins that were identified across 27 samples from three different rumen fractions of three Jersey cows, fed rotationally with three different diets. The functional and structural data of the metaproteomic analysis was further flanked by 16S rRNA gene-based analyses of the archaeal and bacterial community structures and the metabolomes of the rumen fluid fractions were quantified by nuclear magnetic resonance. So far, to the best of our knowledge, there are no studies investigating the metaproteome expressed by the entirety of archaeal and bacterial communities in the different phases of the rumen ecosystem under varying dietary influence. Dietary treatments revealed significant variations in the metaproteome composition and community structures of ruminal bacteria. Host-related effects were not significant. In conclusion, within this project the application of shotgun metaproteomics to characterize the prokaryotic rumen metaproteome was successfully implemented and the obtained results clearly emphasized the benefits of using complementary, state of the art methods to study the microbiome of complex ecosystems like the rumen. Considering the specific functional niches of the rumen microbiome have been shown to be of great importance.
Na+-coupled respiration and reshaping of extracellular polysaccharide layer counteract monensin-induced cation permeability in Prevotella bryantii B14
(2021) Trautmann, Andrej; Schleicher, Lena; Pfirrmann, Jana; Boldt, Christin; Steuber, Julia; Seifert, Jana
Monensin is an ionophore for monovalent cations, which is frequently used to prevent ketosis and to enhance performance in dairy cows. Studies have shown the rumen bacteria Prevotella bryantii B14 being less affected by monensin. The present study aimed to reveal more information about the respective molecular mechanisms in P. bryantii, as there is still a lack of knowledge about defense mechanisms against monensin. Cell growth experiments applying increasing concentrations of monensin and incubations up to 72 h were done. Harvested cells were used for label-free quantitative proteomics, enzyme activity measurements, quantification of intracellular sodium and extracellular glucose concentrations and fluorescence microscopy. Our findings confirmed an active cell growth and fermentation activity of P. bryantii B14 despite monensin concentrations up to 60 µM. An elevated abundance and activity of the Na+-translocating NADH:quinone oxidoreductase counteracted sodium influx caused by monensin. Cell membranes and extracellular polysaccharides were highly influenced by monensin indicated by a reduced number of outer membrane proteins, an increased number of certain glucoside hydrolases and an elevated concentration of extracellular glucose. Thus, a reconstruction of extracellular polysaccharides in P. bryantii in response to monensin is proposed, which is expected to have a negative impact on the substrate binding capacities of this rumen bacterium.
Novel bacterial species from the chicken gastrointestinal tract and their functional diversity
(2023) Rios Galicia, Bibiana; Seifert, Jana
The digestive system of chicken presents different physicochemical conditions along the gastrointestinal tract (GIT), shaping an individual microbial profile along sections with different metabolic capacities and divergence on the adaptations to the environment. Efforts to obtain cultivable bacteria originating from the upper region of chicken GIT enrich the reference genome database and provide information about the site- specific adaptations of bacteria colonizing such GIT sections allowing to understand the metabolic profile and adaptive strategies to the environment. However, the lack of sufficient reference genomes limits the interpretation of sequencing data and restrain the study of complex functions. In this study, 43 strains obtained from crop, jejunum and ileum of chicken were isolated, characterised and genome analysed to observe their metabolic profiles, adaptive strategies and to serve as future references. Eight isolates represent new species that colonise the upper gut intestinal tract and present consistent adaptations that enable us to predict their ecological role, expanding our knowledge on the adaptative functions. Strains of Limosilactobacillus were found to be more abundant in the crop, while Ligilactobacillus dominated the ileal digesta. Isolates from crop encode a high number of glycosidases specialised in complex polysaccharides compared to strains isolated from jejunum and ileum. While isolates from jejunum and ileum encode a higher number of genes that interact with the host such as collagenases and hyaluronidases, indicating preferential persistence and adaptations along the GIT. These results represent the first repository of bacteria obtained from the crop and small intestine of chicken using culturomics, improving the potential handling of chicken microbiome with biotechnological applications
A shift towards succinate‐producing Prevotella in the ruminal microbiome challenged with monensin
(2022) Trautmann, Andrej; Schleicher, Lena; Koch, Ariane; Günther, Johannes; Steuber, Julia; Seifert, Jana
The time‐resolved impact of monensin on the active rumen microbiome was studied in a rumen‐simulating technique (Rusitec) with metaproteomic and metabolomic approaches. Monensin treatment caused a decreased fibre degradation potential that was observed by the reduced abundance of proteins assigned to fibrolytic bacteria and glycoside hydrolases, sugar transporters and carbohydrate metabolism. Decreased proteolytic activities resulted in reduced amounts of ammonium as well as branched‐chain fatty acids. The family Prevotellaceae exhibited increased resilience in the presence of monensin, with a switch of the metabolism from acetate to succinate production. Prevotella species harbour a membrane‐bound electron transfer complex, which drives the reduction of fumarate to succinate, which is the substrate for propionate production in the rumen habitat. Besides the increased succinate production, a concomitant depletion of methane concentration was observed upon monensin exposure. Our study demonstrates that Prevotella sp. shifts its metabolism successfully in response to monensin exposure and Prevotellaceae represents the key bacterial family stabilizing the rumen microbiota during exposure to monensin.
The intestinal microbiome and metabolome of dairy cows under challenging conditions
(2022) Tröscher-Mußotter, Johanna; Seifert, Jana
The modern dairy cow is confronted with a multitude of stressors throughout live. Especially calving, transition, and microbial infections are strong challenges that can have long-lasting impacts on the cow’s health and performance. Yet, individuals can differ in their response towards these challenges, raising the question which characteristics in the dairy cow contribute to a more or less robust animal. Apart from genetics, the gut microbiome and the entailed metabolome is assumed to play an important role in buffering or promoting host stress. This is also due to the fact that the gut microbiome is strongly involved in the hosts energy metabolism and immune system. As dairy cows often show performance impairments during high energy demanding periods, it could be suggested that improving energy metabolism in these specific phases might reduce the negative phenotypic outcomes. This was tested using dietary L-carnitine, a metabolite inevitably necessary for energy metabolism. However, no supplement effects on the intestinal microbiome or metabolome have been found in the present work. Supplementation was continued throughout the complete trial. Calving functioned as an individual stimulus, and an intra-venous LPS injection induced a standardized inflammatory challenge, as a specific amount of LPS per kg of bodyweight was applied per cow. Supplemented animals were compared to a control group. In total, the animals were studied across 168 days and sampled extensively at several sites. The focus of this thesis was to analyze the bacterial consortia and metabolites of both, host and bacteria, in rumen, duodenum, and feces throughout the given period. This was to elucidate the metabolic reactions and bacterial shifts during the mentioned challenging periods and their response to the L-carnitine supplementation. First, the ruminal and duodenal fluid microbiome of eight double cannulated animals during the two respective challenges was analysed. Before calving and feed change, rumen and duodenal fluid bacterial consortia were significantly different, thereafter very alike. Strong microbial community shifts were observed throughout the complete trial irrespectively of the matrix. Both matrices varied in their metabolite patterns indicating functional variation among sites. Also, a strong increase of Bifidobacterium at three days after calving was observed in almost all animals pointing towards a strong biological purpose. This needs to be investigated in upcoming studies. The study could show increasing ketogenic activities in the animals after calving and proposes a possible protective host-microbial interaction, against a ruminal collapse induced by LPS challenge, here described as "microbial airbag". The second part included fecal samples of the same animals, which were analyzed for their bacterial consortia and targeted metabolites. Different dynamics and diversities of microbial communities amongst the individuals were observed, according to which animals could be grouped into three microbiome clusters. These showed in part fundamentally different metabolic, health, and performance parameters, indicating strong host-microbiome-metabolite interactions. The study demonstrated that microbiome clustering may contribute to identifying different metabo- and production types. Again, the study observed a strong increase of Bifidobacterium at three days after calving and even during the LPS challenge supporting the findings of the former study. This strengthens the hypothesis that also for the cow Bifidobacterium may have protective effects, as this genus is largely involved in health promoting activities. The power of this project lies in the massive sampling of different body sites in dairy cows across a very long period of time and finally, merging of the collected data. This, however, requires high computational efforts as numerous time points, matrices, animals, measurements, treatments, feeding regimen, and challenges resulted into a large bandwidth of parameters and metadata. Yet, it bears the potential to better elucidate and understand actions and reactions of the host, its microbiome and metabolism, as well as organ-axes in dairy cows and thereby gaining a more holistic picture of these complex animals. The aim of analyzing the host, its microbiome and metabolome throughout challenging periods resulted into the following main findings. Time, calving, and feed change remarkably change the microbial communities and to a lesser extent the metabolomes in all three matrices. Rumen and proximal duodenal fluid samples significantly differ in their metabolomes but not in their microbiome. In all matrices, an increase of Bifidobacterium is seen within three days after calving, which has to be further researched. Across the herd, three distinct microbiome clusters are found, which significantly differ in their production and health parameters.
The porcine intestinal microbiota : studies on diversity and dietary impact
(2018) Burbach, Katharina; Seifert, Jana
The entirety of microbial communities within the gastrointestinal tract is referred to as intestinal microbiota and is predominantly composed of bacteria. Interactions between the microbiota, the host and the diet are essential for maintaining a healthy and functional intestinal ecosystem. The overarching aim of this thesis was the characterization of the porcine intestinal microbiota and further to enhance knowledge about the effects of varying diets. High-throughput sequencing of the 16S rRNA gene facilitates exploration of the taxonomic composition of the microbiota. However, the respective findings may be impaired by methodological variations. Thus, within this thesis, commercial DNA extraction kits are evaluated for their suitability in porcine microbiota analysis. The tested extractions yield into variations of quantity and quality of DNA. The DNA extracts are further used to elucidate the structure of the microbiota by a rapid fingerprinting (Terminal Restriction Fragment Length Polymorphism) and high-resolution sequencing (Illumina amplicon sequencing). While different variable regions of the 16S rRNA gene vary in the taxonomical resolution, sequencing analyses exhibit a good comparability of the two regions V1-V2 and the V5-V6. Furthermore, the microbiota profiles reveal a high consistency by the fingerprinting and sequencing approach but are distinguished by the different DNA extraction kits. Based on criteria of DNA extraction and the depicted microbiota composition, it is recommended to use the FastDNA SPIN Kit for Soil for further analysis of porcine intestinal microbiota. Subsequently, these methodological findings are applied to investigate the impact of varying diets. Illumina amplicon sequencing of the V1-V2 region of the 16S rRNA gene reveals different microbiota structures when diets are solely composed of rye or triticale. Besides the taxonomic analyses of ileal digesta and fecal samples, the concentrations of bacterial metabolites in feces are determined. In summary, rye promotes an increased abundance of saccharolytic bacteria like Lactobacillus, Bifidobacterium, and Prevotella and results in higher concentrations of bacterial metabolites in fecal samples. In contrast, a diet based on triticale is associated with an increased abundance of Clostridium sensu stricto, which may indicate an enhanced cellulolytic potential of the microbiota. When the crude protein content is increased (18%), compared to a lower content (14%), an increased abundance of Lactobacillus is demonstrated in microbiota of ileal digesta samples. However, the content of crude protein did not affect the overall microbiota significantly. In addition, dietary supplementation with probiotic Bacillus spp. shows no effect. In conclusion, these dietary effects on microbiota are considered together with results of a protein digestibility analysis. Moreover, an impact of dietary calcium and phosphorus in combination with different sources of dietary protein is analyzed by fingerprinting approach of digesta samples. Here, the content of calcium-phosphorus shows significant effects on the microbiota of caecal digesta and the putative identities of discriminative variables are determined by a cloning-sequencing approach. Similar, 16S rRNA gene sequencing reveals a significant impact of dietary calcium-phosphorus on the overall fecal microbiota without indicating specific discriminating variables. In combination with the results of a meta-proteomic approach, a gradual adaptation on dietary changes is indicated and consequently, a prolonged adaptation time of three to four weeks is recommended for diet-microbiota studies. This thesis includes a comprehensive analysis of the microbiota across and along the gastrointestinal tract of piglets and explores the dietary inclusion of four levels of insect larvae meal. Feeding insects represent an alternative source of dietary protein, whereby the increased content of chitin indicates a potential shift in microbiota composition compared to a control diet. However, in this case, the structural analysis demonstrates no effects on the overall microbiota’s structure. However, a pairwise comparison between diets reveals significant effects on the microbiota of digesta samples of the small intestine. Dietary inclusion of 5% insect meal increases the abundance of Lactobacillus, whereas the control treatment promotes Bifidobacterium. In conclusion, the results of the present thesis emphasize the importance of standardization within 16S rRNA gene based studies of the porcine intestinal microbiota. Furthermore, the necessity of studying various sampling sites combined with multidisciplinary approaches is demonstrated.
Treatment of benzene and ammonium contaminated groundwater using microbial electrochemical technology and constructed wetlands
(2018) Wei, Manman; Seifert, Jana
With the rapid development of modern industry, energy shortage and environmental pollution are getting more and more serious. Groundwater pollution is one of the most important problems. A multitude of remediation techniques in situ or ex situ have been used to treat contaminated groundwater. This thesis was to investigate whether groundwater contaminated mainly by benzene and ammonium can be remediated by constructed wetlands in combination with microbial electrochemical technology. The objectives of this thesis are (i) to develop and test systems for removing pollutants and simultaneously recovering energy from contaminated groundwater, (ii) to maximize the benefits of both constructed wetland and microbial electrochemical technology while treating contaminated groundwater, (iii) to elucidate the underlying electrochemical reactions and pollutant degradation pathways, and (iv) to investigate microbial active species and functional proteins involved in benzene degradation and ammonium removal. A microbial fuel cell (MFC) equipped with an aerated cathode and a control without aeration at the cathode were designed to remove benzene and ammonium from contaminated groundwater collected in the Leuna site (Saxony-Anhalt, Germany). The performance of pollutant removal and electricity generation was investigated and compared in the two reactors. Electrochemical processes occurring in the MFC were determined by benzene and ammonium spiking experiments as well as oxygen interruption experiments. Additionally, the biodegradation pathways and dominant organisms were elucidated by compound specific stable isotope analysis (CSIA) and Illumina sequencing. The results indicated the principal feasibility of treating benzene and ammonium contaminated groundwater by a MFC equipped with an aerated cathode. Benzene (~15 mg/L) was completely removed in the MFC, of which 80% disappeared already at the anoxic anode. Ammonium (~20 mg/L) was oxidized to nitrate at the cathode; this reaction was not directly linked to electricity generation. The maximum power density was 316 mW/m3 net anoxic compartment (NAC) at a current density of 0.99 A/m3 NAC. Coulombic and energy efficiencies of 14% and 4% were obtained based on the anodic benzene degradation. Benzene was initially activated by enzymatic monohydroxylation at the oxygen-limited anode; the further anaerobic oxidation of the intermediate metabolites released electrons accompanied by electrons transfer to the anode. Dominant phylotypes at the MFC anode revealed by 16S rRNA Illumina sequencing were affiliated to the Chlorobiaceae, Rhodocyclaceae and Comamonadaceae, presumably associated with benzene degradation. Nitrification took place at the aerated cathode of the MFC and was catalyzed by phylotypes belonging to the Nitrosomonadales and Nitrospirales. The control reactor failed to generate electricity, although phylotypes affiliated to the Chlorobiaceae, Rhodocyclaceae and Comamonadaceae were dominant as well; the control reactor can be thus regarded as a mesocosm in which granular graphite was colonized by benzene degraders, but showed a lower benzene removal efficiency compared to the MFC. In order to enhance benzene and ammonium removal while simultaneously harvesting energy, a constructed wetland integrated with microbial electrochemical technology (MET-CW) was established by embedding four anode modules into the sand bed and connecting it to a cathode placed in the open pond inside a bench-scale horizontal subsurface flow constructed wetland (HSSF-CW). Compared with the control CW, enhanced benzene and ammonium removal efficiencies were found in the MET-CW. The electrochemical performances of anode modules located at the four different depths were compared; the results showed that anode modules located in the deep layer (Module 3 and 4) had the relatively high power densities whereas the power densities located in the upper layer (modules 1 and 2) were extremely low. The initial activity mechanism of benzene degradation was analyzed by CSIA. Ammonium removal processes were assessed using nitrogen isotope fractionation of ammonium. Functional proteins and active microbial species involved in nitrogen transformation processes were detected using protein-based stable isotope probing (protein-SIP) with in situ feeding of 15N-NH4+. Additionally, potential denitrification and anammox rates were measured using Nitrogen isotope tracing. The results demonstrated that benzene and ammonium removal in a CW can be improved by combination with microbial electrochemical technology. The enhanced benzene removal was linked to the use of the anode modules as electron acceptor, whereas efficient ammonium removal was probably attributed to the elimination of inhibition effects by the co-contaminant benzene. Benzene was initially activated by monohydroxylation, forming intermediates which were subsequently oxidized accompanied by extracellular electron transfer, leading to current production. Partial nitrification accompanied by either heterotrophic denitrification or nitrifier-denitrification was mainly responsible for NH4+-N removal in the MET-CW, whereas anammox played a minor role. However, the contribution of anammox was markedly increased at the location near to the anode modules. In summary, this research indicated that microbial electrochemical technology can be used to improve the performance of pollutant removal while simultaneously harvesting energy from contaminated groundwater. Especially, the combination of MET with other traditional treatment approaches (e.g. constructed wetland) is a promising alternative to treat contaminated water.
Untersuchung der Energie- und Nährstoffflüsse mikrobieller Gemeinschaften
(2017) Starke, Robert; Seifert, Jana
The activity of microorganisms was heavily investigated using the incorporation of stabile isotopes in the last decade. Here, all biomolecules but predominantly DNA, RNA, proteins and phospholipid derived fatty acids are used to trace the label in the biomass of active microbes. Thereby, the phylogenetic information decreases from DNA and RNA to proteins whereas the latter allow to describe the actual phenotype. In this work, protein stable isotope probing (protein-SIP) was applied to two different microbial systems: (a) the anaerobic mineralization of benzene and (b) the assimilation of plant-derived organic matter in soil. Labeling of the secondary metabolism of the benzene-mineralizing and sulfate-reducing community using 13C2-acetate: The well-described microbial community enriched from the Zeitz aquifer was fed with the postulated and fully 13C-labeled intermediate of syntrophic benzene fermentation, acetate, to unveil detailed secondary utilization processes. Additional acetate amended to the ongoing benzene mineralization showed no influence on sulfide produced by sulfate reduction. Instead, labeled acetate was incorporated by Campylobacterales, Syntrophobacterales, Archaeoglobales, Clostridiales and Desulfobacterales in descending order. The epsilonproteobacterial Campylobacterales featured the fastest and the highest 13C-incorporation to confirm previous metagenome-based studies and to assign a physiological role to this phylotype of the community for the first time. Metagenome based labeling of the secondary metabolism of the benzene-mineralizing and sulfate-reducing community: In this study, the population genome of the primary acetate utilizer was reconstructed from the metagenome of the benzene mineralizing community obtained by whole-genome shotgun sequencing. Genomic DNA originated from a starvation enrichment culture previously metabolizing m-xylen and enriched in the identical epsilonproteobacterial phylotype of this community. The presence of the sulfide quinone oxidoreductase (sqr) and the polysulfide reductase (psr) suggested a key role in sulfur cycling. Hence, the epsilonproteobacterial phylotype is able to oxidize otherwise toxic sulfid produced by sulfate reduction to polysulfide via SQR and its subsequent reduction to sulfide via PSR. Further, the detection of an acetate transporter (actP) and the acetyl-CoA synthetase (acsA) for acetate activation approved direct assimilation as shown in the previous study. Short-term assimilation of plant-derived organic matter in soil: In this protein-SIP study, the short-term assimilation of plant-derived organic matter in soil was demonstrated using 15N-labeled tobacco for the first time. In contrast to the postulated model in which fungi degrade plant-derived complex compounds and secrete low molecular weight compounds which are then degraded by bacteria, our study demonstrated the dominance of bacteria over fungi during the short-term assimilation of plant-derived organic matter. Bacteria outcompete fungi for the easy available plant-derived compounds until complex compounds such as cellulose and lignin are enriched and degraded by slow growing fungi. The use of multiOMIC techniques resulted in a multidimensional scheme to easily group and categorize different behaviours of microorganisms.