A Robust and Universal Metaproteomics Workflow for Research Studies and Routine Diagnostics Within 24 h Using Phenol Extraction, FASP Digest, and the MetaProteomeAnalyzer

The investigation of microbial proteins by mass spectrometry (metaproteomics) is a key technology for simultaneously assessing the taxonomic composition and the functionality of microbial communities in medical, environmental, and biotechnological applications. We present an improved metaproteomics workflow using an updated sample preparation and a new version of the MetaProteomeAnalyzer software for data analysis. High resolution by multidimensional separation (GeLC, MudPIT) was sacrificed to aim at fast analysis of a broad range of different samples in less than 24 h. The improved workflow generated at least two times as many protein identifications than our previous workflow, and a drastic increase of taxonomic and functional annotations. Improvements of all aspects of the workflow, particularly the speed, are first steps toward potential routine clinical diagnostics (i.e., fecal samples) and analysis of technical and environmental samples. The MetaProteomeAnalyzer is provided to the scientific community as a central remote server solution at www.mpa.ovgu.de.Peer Reviewe

Metaproteome analysis reveals that syntrophy, competition, and phage-host interaction shape microbial communities in biogas plants

Background: In biogas plants, complex microbial communities produce methane and carbon dioxide by anaerobic digestion of biomass. For the characterization of the microbial functional networks, samples of 11 reactors were analyzed using a high-resolution metaproteomics pipeline. Results: Examined methanogenesis archaeal communities were either mixotrophic or strictly hydrogenotrophic in syntrophy with bacterial acetate oxidizers. Mapping of identified metaproteins with process steps described by the Anaerobic Digestion Model 1 confirmed its main assumptions and also proposed some extensions such as syntrophic acetate oxidation or fermentation of alcohols. Results indicate that the microbial communities were shaped by syntrophy as well as competition and phage-host interactions causing cell lysis. For the families Bacillaceae, Enterobacteriaceae, and Clostridiaceae, the number of phages exceeded up to 20-fold the number of host cells. Conclusion: Phage-induced cell lysis might slow down the conversion of substrates to biogas, though, it could support the growth of auxotrophic microbes by cycling of nutrients. © 2019 The Author(s)

Resource recovery from the anaerobic digestion of food waste is underpinned by cross-kingdom microbial activities

This work was supported by the Earth and Natural Sciences (ENS) Doctoral Studies Programme, funded by the Higher Education Authority (HEA) of Ireland through the Programme for Research at Third Level Institutions, Cycle 5 (PRTLI-5), co-funded by the European Regional Development Fund (ERDF).As the human population grows on the planet so does the generation of waste and particularly that of food waste. In order to tackle the world sustainability crisis, efforts to recover products from waste are critical. Here, we anaerobically recovered volatile fatty acids (VFAs) from food waste and analysed the microbial populations underpinning the process. An increased contribution of fungi relative to bacteria was observed throughout the reactor operation, with both kingdoms implicated into the main three steps of anaerobic digestion occurring within our systems: hydrolysis, acidogenesis and acetogenesis. Overall, Ascomycota, Proteobacteria and Firmicutes were found to drive the anaerobic digestion of food waste, with butyrate as the most abundant VFA likely produced by Clostridium using lactate as a precursor. Taken together we demonstrate that the generation of products of added-value from food waste results from cross-kingdoms microbial activities implicating fungi and bacteria.Publisher PDFPeer reviewe

Assessing the impact of interfering organic matter on soil metaproteomic workflow

Funding: Matthias Waibel was funded by the University of Galway College of Science and the Irish Research Council under GOIPG/2016/1215. The James Hutton Institute receives funding support from the Rural and Environment Science and Analytical Services Division of the Scottish Government. Open access funding provided by IReL.Soil organic matter (SOM) is biologically, chemically, and physically complex. As a major store of nutrients within soil, it plays an important role in nutrient provision to plants. An enhanced understanding of SOM utilisation processes could underpin better fertiliser management for plant growth, with reduced environmental losses. Metaproteomics can allow the characterisation of protein profiles and could help gaining insights into SOM microbial decomposition mechanisms. Here, we applied three different extraction methods to two soil types to recover SOM with different characteristics. Specifically, water extractable organic matter, mineral associated organic matter and protein-bound organic matter were targeted with the aim to investigate the metaproteome enriched in those extractions. As a proof-of-concept replicated extracts from one soil were further analysed for peptide identification using liquid chromatography followed by tandem mass spectrometry. We employ a framework for mining mass spectra for both peptide assignment and fragmentation pattern characterisation. Different extracts were found to exhibit contrasting total protein and humic substance content for the two soils investigated. Overall, water extracts displayed the lowest humic substance content (in both soils) and the highest number of peptide identifications (in the soil investigated) with most frequent peptide hits associated with diverse substrate/ligand binding proteins of Proteobacteria and derived taxa. Our framework also highlighted a strong peptidic signal in unassigned and unmatched spectra, information that is currently not captured by the pipelines employed in this study. Taken together, this work points to specific areas for optimisation in chromatography and mass spectrometry to adequately characterise SOM associated metaproteomes.Publisher PDFPeer reviewe

Diversity, specificity and evolutionary history of marine invertebrate symbioses and functions of the sulfur-oxidizing symbionts

Many marine invertebrates have established symbioses with chemosynthetic bacteria that metabolize reduced sulfur compounds and provide nutrition to their host. Two key questions in the field of symbiosis are: (1) How specific and evolutionarily stable are these symbioses? Chapters II, III and IV of this thesis contribute to a more comprehensive understanding of this question by investigating the diversity, specificity and evolutionary history of three sulfur-oxidizing symbioses: deep-sea vestimentiferan tubeworm endosymbioses, shallow water gutless phallodriline oligochaete endo- and stilbonematine nematode ectosymbioses. The studies emphasize the power of molecular analyses to uncover hidden symbiont diversity and highlight the remarkably stable and specific stilbonematine ectosymbioses. (2) What are the benefits for the symbiotic partners? Chapter V strengthens the hypothesis of stilbonematine ectosymbionts' nutritional role for their host and the genomic study in Chapter VI discusses potential additional functions of the ectosymbionts for their nematode host

Analyse comparative de la prévalence et de la diversité des communautés bactériennes des ensilages et du lait cru bovin

Le microbiote du lait cru est un déterminant majeur de sa qualité et de celle des produits dérivés. Dans les fermes laitières, l'hygiène et la santé du pis, les fèces, la litière et les fourrages conservés sont les principales sources de microorganismes qui contaminent l'environnement de l'étable et qui peuvent se retrouver dans le lait. Les ensilages de légumineuses et de graminées produits par fermentation lactique, outre les bactéries lactiques, sont riches en espèces microbiennes dont la prévalence, la diversité et l'abondance dépendent de facteurs incontrôlables tels que les paramètres environnementaux, et de facteurs contrôlables tels que les pratiques de gestion adoptées par les fermiers. Le choix du type de fourrages parmi lesquels le foin, les ensilages d'herbe/légume ou de maïs, l'utilisation ou non d'inoculants, le type d'inoculant commercial utilisé et les types de structures d'entreposage sont autant d'éléments qui influencent la composition microbienne des ensilages et dont la gestion a un impact peu documenté sur la qualité microbiologique du lait cru. Les travaux de cette thèse portent sur l'écologie microbienne des fourrages préservés et l'évaluation de leur contribution à la contamination du lait cru de vache. Pour ce faire, des méthodes de préservation d'échantillons de lait cru à base d'azidiol ou de bronopol ont été évaluées pour leur capacité à maintenir intactes la viabilité et l'abondance des communautés bactériennes présentes. Des échantillons de foin, d'ensilages inoculés et non inoculés, et de lait cru ont été prélevés deux fois, à l'automne 2015 et au printemps 2016 dans 24 fermes laitières au Québec. Les analyses métataxonomiques et des charges microbiennes déterminées par PCR quantitative après le traitement ou non des cellules microbiennes au propidium monoazide ont montré que l'azidiol combiné au diméthyle sulfoxide et une température de -20 °C permet de stabiliser le microbiote du lait cru pendant au moins 30 jours, et que l'azidiol seul maintient l'intégrité des communautés bactériennes pendant 10 jours à 4 °C. Ces études ont également démontré que le séquençage à haut-débit des régions V3-V4 et V6-V8 du gène codant pour l'ARN ribosomique 16S génère des données dont l'exploitation peut conduire à des conclusions plus ou moins divergentes selon les hypothèses de départ. L'importance d'un choix judicieux de la région hyper variable à séquencer est soulignée. Nos résultats ont révélé des différences entre le microbiote du foin et celui des ensilages, ainsi qu'entre les types d'ensilage inoculés et non inoculés. De plus, les rations alimentaires à base de foin, d'ensilage d'herbe/légume non inoculé, d'un mélange d'ensilage d'herbe/légume non inoculé et d'ensilage de maïs inoculé, ou d'un mélange d'ensilage d'herbe/légume et de maïs inoculés et non inoculés partagent jusqu'à 31 % de leur microbiote identifié au niveau de variants de séquences avec le lait cru produit dans les fermes correspondantes. Les taxons plausiblement transférés des fourrages au lait appartiennent surtout aux Proteobacteria, Firmicutes et Actinobacteria. Les résultats obtenus suggèrent que la contamination bactérienne du lait cru par les fourrages se fait de manière aléatoire. Il ressort de nos études que ces taxons supposément transférés des ensilages sont en grande partie responsables des différences observées entre les communautés bactériennes du lait des cinq types de rations. Bien que les structures phylogénétiques des échantillons de lait produits par les vaches alimentées avec les rations d'ensilages non inoculés ou inoculés se soient montrées significativement différentes, il est difficile de conclure sur l'impact réel des inoculants sur la qualité microbiologique du lait cru. L'analyse des réseaux de co-occurrence et de co-exclusion au sein du microbiote a révélé d'une part, dans les ensilages les interactions entre les bactéries lactiques et non-lactiques qui pourraient considérablement influencer le processus de fermentation et ultimement la qualité du produit final, et d'autre part, dans le lait des niches microbiennes associées aux sites de contamination du lait dans l'environnement à la ferme. Par l'implémentation des méthodes d'analyses multivariées et multi-table intégrant le microbiote et les paramètres physico-chimiques des matrices alimentaires échantillonnées, cette thèse propose une approche d'exploitation des données de métataxonomique permettant d'approfondir nos connaissances de la microbiologie du lait cru et des produits laitiers.The microbiota of raw bovine milk is tightly associated with its quality and that of derivatives. On dairy farms, udder health, faeces, beddings, and fermented forage are among the main sources of milk microbial contaminants. Grass/legume and corn silage obtained by lactic fermentation harbour complex microbial community of which the diversity, the prevalence, and abundance are driven by uncontrollable factors such as environmental conditions, or controllable factors inherent to farm management practices. Forage types including hay, grass/legume or corn silage, the use of microbial additives and their commercial types, and the types of storage structures may influence community assembly of mature silage. However, the impact of forage management practices on the raw milk microbiota is not fully understood. This thesis aimed at investigating the microbial ecology of preserved forage and assessing their contribution to the raw milk contamination. To do so, the ability of milk sample preservation methods based on azidiol or bronopol to maintain viable and stable microbiota over time was assessed. Forage and raw milk samples were collected twice from 24 dairy farms in Quebec, in the fall 2015 and the spring 2016. Analyses of metataxonomic and qPCR-based bacterial load data derived from cells treated or not with propidium monoazide to account for viability showed that azidiol combined with dimethyl sulfoxide prevented the microbiota instability of raw milk stored at -20 °C for at least 30 days. Azidiol used alone was additionally found to keep the microbiota in milk samples intact for up to 10 days when stored at 4 °C. It was demonstrated that for hypothesis-driven microbiota studies, divergent conclusions can be drawn from hight-throughput sequencing of the V3-V4 and V6-V8 hypervariable regions of the 16S rRNA gene pool. The importance of the hypervariable region to target is therefore highlighted. Our study revealed differences between hay microbiota and that of silage, whether inoculated or not. Moreover, forage ration combinations shared up to 31 % of their bacterial phylotypes with raw milk samples produced in the corresponding farms. Taxa that were probably transferred from forage ration combinations to raw milk encompassed the phyla Proteobacteria, Firmicutes, and Actinobacteria. Our results suggested that raw milk contamination on the farm occurs erratically, and that transferred taxa were mainly involved in differential abundance outcomes. Although the microbiota of milk samples associated with the five forage ration combinations exhibited differences in phylogenetic composition, concluding on the effects of microbial additives used for ensiling is a challenge. In this thesis, the analysis of bacteria interaction networks showed that co-occurrence or co-exclusion of lactic and non-lactic bacteria might considerably affect the microbiological quality of mature silage at feed-out. On the other hand, the same analysis performed with milk samples revealed microbial niches associated with milk contamination points on the farm environment. Through the implementation of multivariate multi-table analysis methods that integrated data from the microbiota and from the physicochemical characteristics of the sampled matrices, this thesis suggests methodological approaches that exploit metataxonomic data to deepen our knowledge of the raw milk microbiota and dairy products

Evaluation of Human Microbiota-Associated (HMA) Porcine Models to Study the Human Gastrointestinal Microbiome

Research conducted in the past couple of decades has showcased the importance of the gut microbiota in human health and well-being. While many studies have reported on the differences in community membership between a disease state and a healthy state, few have investigated the mechanisms through which an aberrant microbiota contributes to a disease phenotype. One of the primary reasons for this are the many technical and ethical barriers to conducting the necessary studies directly in human individuals. Human microbiota-associated (HMA) porcine models have the potential to become important research tools which can enable the testing of hypotheses regarding host-microbiota interactions in human health and disease without directly involving humans. However, relatively few microbiome studies have utilized porcine models in this capacity. Through multiple studies, we evaluated HMA porcine models in terms of their suitability for use in gut microbiota studies. Results demonstrated that (1) compared to an HMA C3H/HeN mouse model, a higher percentage of donor taxa from donors of different age groups were able to persistently colonize HMA piglets, (2) while a majority of donor taxa in infant donors were able to colonize HMA piglets, rare/low-abundance taxa found in the infant donors enriched once engrafted into the piglets, and (3) the potential for using HMA piglets for studying host-microbiota interactions related to obesity. We believe that further improvements to address some of the shortcoming and challenges associated with HMA piglets will facilitate more wide-spread use of this animal model in the field of gut microbiome research. Advisor: Samodha C. Fernand