Challenges, Strategies, and Perspectives for Reference-Independent Longitudinal Multi-Omic Microbiome Studies

in Frontiers in Genetics (2021)

Integration of omics data for biotechnology-relevant microbial communities

Doctoral thesis (2018)

Naturally occurring and artificial bacterial communities play an import role in many biotechnological processes. To elucidate bacterial interactions that are important for potential optimized ... [more ▼]

Naturally occurring and artificial bacterial communities play an import role in many biotechnological processes. To elucidate bacterial interactions that are important for potential optimized biotechnological applications, high-throughput measurements of biomolecules, metagenomics, metratranscriptomics,metaproteomics, and meta-metabolomics provide a detailed snapshot of mixed microbial consortia. Integration of multiple layers of omics data allows to reconstruct structure and function of complex microbial communities and is demonstrated for two different model systems. The first chapter focuses on synthetic communities consisting of strains representing key species found in biomining operations and acid mine drainage and that are of economical interest for copper production. A high-quality closed reference genome for L. ferriphilum was obtained by DNA sequencing and was subsequently used to integrate functional omics data, i.e. transcriptomic and proteomic profiling. The combination of genomics, genome annotation, and functional omics data allowed an in-depth characterization of L. ferriphilum in culture medium and in the presence of the iron sulfide mineral chalcopyrite, an economically relevant copper ore. Subsequently, analyses were performed for co-cultures of up to three organisms highlighting specific interaction mechanisms. The cultures without L. ferriphilum showed higher copper solubilisation rates, as the highly efficient iron oxidiser might raise the redox potential above the optimal range. For in situ studies, reference-based analyses are of limited use, e.g. due to a lack in reference genomes of culturable isolates. Hence, the second chapter focuses on an approach to study mixed microbial communities independent of prior knowledge and available reference genomes. A timeseries of oleaginous floating sludge samples that spans over one and a half years was analysed by integrating metagenomic, metatranscriptomic, metaproteomic, and meta-metabolomic data. This allowed the reconstruction of population level genomes and the characterization of the niches of the respective populations. The functional potential was assessed, as well as expression profiles over time, yielding a detailed view on lifestyle strategies and the potential impact of abiotic factors. Understanding the niche ecology of the predominant lipid accumulators in the system could lead towards optimized biofuel production. [less ▲]

Microbiome-derived Multi-omicBiomarkers for Early-stage Detection and Stratification of Parkinson's Disease

Poster (2018, August)

Weak Iron Oxidation by Sulfobacillus thermosulfidooxidans Maintains a Favorable Redox Potential for Chalcopyrite Bioleaching

in Frontiers in Microbiology (2018), 9(December), 1-12

Bioleaching is an emerging technology, describing the microbially assisted dissolution of sulfidic ores that provides a more environmentally friendly alternative to many traditional metal extraction ... [more ▼]

Bioleaching is an emerging technology, describing the microbially assisted dissolution of sulfidic ores that provides a more environmentally friendly alternative to many traditional metal extraction methods, such as roasting or smelting. Industrial interest is steadily increasing and today, circa 15–20% of the world’s copper production can be traced back to this method. However, bioleaching of the world’s most abundant copper mineral chalcopyrite suffers from low dissolution rates, often attributed to passivating layers, which need to be overcome to use this technology to its full potential. To prevent these passivating layers from forming, leaching needs to occur at a low oxidation/reduction potential (ORP), but chemical redox control in bioleaching heaps is difficult and costly. As an alternative, selected weak iron-oxidizers could be employed that are incapable of scavenging exceedingly low concentrations of iron and therefore, raise the ORP just above the onset of bioleaching, but not high enough to allow for the occurrence of passivation. In this study, we report that microbial iron oxidation by Sulfobacillus thermosulfidooxidans meets these specifications. Chalcopyrite concentrate bioleaching experiments with S. thermosulfidooxidans as the sole iron oxidizer exhibited significantly lower redox potentials and higher release of copper compared to communities containing the strong iron oxidizer Leptospirillum ferriphilum. Transcriptomic response to single and co-culture of these two iron oxidizers was studied and revealed a greatly decreased number of mRNA transcripts ascribed to iron oxidation in S. thermosulfidooxidans when cultured in the presence of L. ferriphilum. This allowed for the identification of genes potentially responsible for S. thermosulfidooxidans’ weaker iron oxidation to be studied in the future, as well as underlined the need for new mechanisms to control the microbial population in bioleaching heaps. [less ▲]

Automated Microscopic Analysis of Metal Sulfide Colonization by Acidophilic Microorganisms.

in Applied and environmental microbiology (2018), 84(20),

Industrial biomining processes are currently focused on metal sulfides and their dissolution, which is catalyzed by acidophilic iron(II)- and/or sulfur-oxidizing microorganisms. Cell attachment on metal ... [more ▼]

Industrial biomining processes are currently focused on metal sulfides and their dissolution, which is catalyzed by acidophilic iron(II)- and/or sulfur-oxidizing microorganisms. Cell attachment on metal sulfides is important for this process. Biofilm formation is necessary for seeding and persistence of the active microbial community in industrial biomining heaps and tank reactors, and it enhances metal release. In this study, we used a method for direct quantification of the mineral-attached cell population on pyrite or chalcopyrite particles in bioleaching experiments by coupling high-throughput, automated epifluorescence microscopy imaging of mineral particles with algorithms for image analysis and cell quantification, thus avoiding human bias in cell counting. The method was validated by quantifying cell attachment on pyrite and chalcopyrite surfaces with axenic cultures of Acidithiobacillus caldus, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans. The method confirmed the high affinity of L. ferriphilum cells to colonize pyrite and chalcopyrite surfaces and indicated that biofilm dispersal occurs in mature pyrite batch cultures of this species. Deep neural networks were also applied to analyze biofilms of different microbial consortia. Recent analysis of the L. ferriphilum genome revealed the presence of a diffusible soluble factor (DSF) family quorum sensing system. The respective signal compounds are known as biofilm dispersal agents. Biofilm dispersal was confirmed to occur in batch cultures of L. ferriphilum and S. thermosulfidooxidans upon the addition of DSF family signal compounds.IMPORTANCE The presented method for the assessment of mineral colonization allows accurate relative comparisons of the microbial colonization of metal sulfide concentrate particles in a time-resolved manner. Quantitative assessment of the mineral colonization development is important for the compilation of improved mathematical models for metal sulfide dissolution. In addition, deep-learning algorithms proved that axenic or mixed cultures of the three species exhibited characteristic biofilm patterns and predicted the biofilm species composition. The method may be extended to the assessment of microbial colonization on other solid particles and may serve in the optimization of bioleaching processes in laboratory scale experiments with industrially relevant metal sulfide concentrates. Furthermore, the method was used to demonstrate that DSF quorum sensing signals directly influence colonization and dissolution of metal sulfides by mineral-oxidizing bacteria, such as L. ferriphilum and S. thermosulfidooxidans. [less ▲]

First draft genome sequence of a strain belonging to the Zoogloea genus and its gene expression in situ

in Standards in Genomic Sciences (2017), 12(64),

The Gram-negative beta-proteobacterium Zoogloea sp. LCSB751 (LMG 29444) was newly isolated from foaming activated sludge of a municipal wastewater treatment plant. Here, we describe its draft genome ... [more ▼]

The Gram-negative beta-proteobacterium Zoogloea sp. LCSB751 (LMG 29444) was newly isolated from foaming activated sludge of a municipal wastewater treatment plant. Here, we describe its draft genome sequence and annotation together with a general physiological and genomic analysis, as the first sequenced representative of the Zoogloea genus. Moreover, Zoogloea sp. gene expression in its environment is described using metatranscriptomic data obtained from the same treatment plant. The presented genomic and transcriptomic information demonstrate a pronounced capacity of this genus to synthesize poly-β-hydroxyalkanoate within wastewater. [less ▲]

Multi-omics reveal the lifestyle of the acidophilic, mineral-oxidizing model species Leptospirillum ferriphilum(T).

in Applied and environmental microbiology (2017)

Leptospirillum ferriphilum plays a major role in acidic, metal rich environments where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as ... [more ▼]

Leptospirillum ferriphilum plays a major role in acidic, metal rich environments where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of this model species' type strain is available, limiting the possibilities to investigate the strategies and adaptations Leptospirillum ferriphilum(T) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilum(T) DSM 14647 obtained by PacBio SMRT long read sequencing for use as a high quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as substrate and bioleaching cultures containing chalcopyrite (CuFeS2). Leptospirillum ferriphilum(T) adaptations to growth on chalcopyrite included a possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, expression and translation of genes responsible for chemotaxis and motility were enhanced.IMPORTANCELeptospirillum ferriphilum is one of the most important iron-oxidizers in the context of acidic and metal rich environments during moderately thermophilic biomining. A high-quality circular genome of Leptospirillum ferriphilum(T) coupled with functional omics data provides new insights into its metabolic properties, such as the novel identification of genes for atmospheric nitrogen fixation, and represents an essential step for further accurate proteomic and transcriptomic investigation of this acidophile model species in the future. Additionally, light is shed on Leptospirillum ferriphilum(T) adaptation strategies to growth on the copper mineral chalcopyrite. This data can be applied to deepen our understanding and optimization of bioleaching and biooxidation, techniques that present sustainable and environmentally friendly alternatives to many traditional methods for metal extraction. [less ▲]

Identification, recovery, and refinement of hitherto undescribed population-level genomes from the human gastrointestinal tract

in Frontiers in Microbiology (2016), 7(884),

Linking taxonomic identity and functional potential at the population-level is important for the study of mixed microbial communities and is greatly facilitated by the availability of microbial reference ... [more ▼]

Linking taxonomic identity and functional potential at the population-level is important for the study of mixed microbial communities and is greatly facilitated by the availability of microbial reference genomes. While the culture-independent recovery of population-level genomes from environmental samples using the binning of metagenomic data has expanded available reference genome catalogs, several microbial lineages remain underrepresented. Here, we present two reference-independent approaches for the identification, recovery, and refinement of hitherto undescribed population-level genomes. The first approach is aimed at genome recovery of varied taxa and involves multi-sample automated binning using CANOPY CLUSTERING complemented by visualization and human-augmented binning using VIZBINpost hoc. The second approach is particularly well-suited for the study of specific taxa and employs VIZBINde novo. Using these approaches, we reconstructed a total of six population-level genomes of distinct and divergent representatives of the Alphaproteobacteria class, the Mollicutes class, the Clostridiales order, and the Melainabacteria class from human gastrointestinal tract-derived metagenomic data. Our results demonstrate that, while automated binning approaches provide great potential for large-scale studies of mixed microbial communities, these approaches should be complemented with informative visualizations because expert-driven inspection and refinements are critical for the recovery of high-quality population-level genomes. [less ▲]