Annual Conference 2025 of the Association for General and Applied Microbiology - Jahrestagung 2025 der Vereinigung für Allgemeine und Angewandte Mikrobiologie

From taxonomy to metabolism: Exploring the Methane Production Potential of Anaerobic Microbiomes

Late Poster

Anaerobic digestion is a sustainable strategy for the treatment of organic waste, notable for its ability to produce biogas and reduce environmental impact. This study addresses the challenges of understanding the complex dynamics of microbial communities in this process using an approach that integrates metagenomic analyses and genome-scale metabolic modelling (GEM). Combinations of inoculants (wastewater treatment plant sludge and cattle manure) with substrates (pig manure, poultry manure and food waste) were evaluated under different conditions and their impact on microbial composition, metabolic dynamics and methane production was analysed.Reconstruction of microbial communities from MAGs (genomes assembled from metagenomes) was a major challenge due to the complexity and diversity of the environmental samples. Nevertheless, high quality genomes were obtained, allowing the characterisation of key methanogenic archaea and fermentative bacteria. These results highlight the importance of syntrophic interactions in methane production and substrate degradation.Metabolic modelling with GEMs facilitated the simulation of processes such as methanogenesis and the production of intermediate metabolites (acetate, lactate and butyrate). The models predicted clear differences in metabolic performance according to inoculum and substrate combinations, showing an increase in methane production under certain co-digestion conditions. Furthermore, temporal analysis revealed how communities evolve to maximise metabolic efficiency throughout the process.This study highlights the importance of reconstructing microbial communities from MAGs as a tool for understanding and optimising anaerobic digestion. The results highlight the critical role of microbial interactions in process efficiency, providing a basis for improving biogas production and designing more sustainable and efficient waste treatment systems.Keywords: Anaerobic digestion, microbial communities, metagenome-assembled genomes (MAGs), genome-scale metabolic models, methane production, Co-digestion, Syntrophic interactions, waste treatment optimization

Poster

Integrating Bioinformatics for Microbial Discovery: Unlocking Polyhydroxyalkanoates (PHA) Potential in Hypersaline Environments

Background and Aim: Petroleum-based plastics contribute to persistent environmental pollution and health risks. Polyhydroxyalkanoates (PHAs) offer a promising biodegradable and biocompatible alternative. This study investigates metagenomic data from hypersaline environments to identify novel metabolic pathways involved in PHA production. Methods: Water and sediment samples were collected from hypersaline ecosystems in southern Tunisia. Oxford Nanopore and Illumina sequencing technologies were used to generate metagenomes. Hidden Markov Model (HMM)-based bioinformatics pipelines were developed to identify PHA biosynthetic gene clusters in halophilic microorganisms. Results: Analysis revealed key genes involved in PHA biosynthesis, including PHA synthases, depolymerases, and regulatory proteins. These genes are central to the polymerization and degradation of PHAs. The identification of shared operon structures indicates that PHA-synthesis is encoded in conserved operon structures. Functional validation through genetic engineering is planned to confirm their roles in PHA production. Conclusion: This study highlights the biosynthetic potential of halophilic microorganisms in hypersaline environments, emphasizing their promise for sustainable bioplastic production. Keywords: Bioplastics, Polyhydroxyalkanoates, Hypersaline Microorganisms, Metagenomics, Hidden Markov Models, Sustainable Alternatives

Prof.

Utilizing photosystem I to drive CYP153A monooxygenase activity by light

Nowadays, the chemical industry has a great interest in the sustainable production of chemicals such as ω-hydroxylated hydrocarbons or epoxides as chemical feedstocks. As an alternative to the chemical synthesis, the enzymatic approach is considered promising. CYP153A oxidoreductases, a family of the P450 monooxygenase superfamily, are known to catalyze diverse oxidation reactions, e.g. the terminal hydroxylation of various carbon hydrates or the epoxidation of carbon double bounds in a highly regio- and stereoselective manner. In situ, CYP153A enzymes are part of a NAD(P)H dependent three component system consisting of a reductase and a ferredoxin as an electron carrier.The accessibility of NAD(P)H as the electron donor is one of the most relevant limitations for the enzymatic biocatalysis. Various NAD(P)H regeneration systems are available but are also harboring unfavorable downsides. A strategy to overcome the dependence of NAD(P)H is the functional coupling of the CYP153A enzymes to the photosynthetic electron transport chain to enable a light dependent biocatalysis. As a proof of concept, a photosystem I (PSI) driven cascade was established, utilizing combinations of two ferredoxins from Thermosynechococcus vestitus and Acinetobacter sp. OC4 as an electron mediator to enable the monooxygenase activity of two CYP153A candidates from Polaromonas sp. JS666 or Gordonia rubripertincta CWB2.Active PSI trimers were isolated from the thermophilic cyanobacterium T. vestitus via hydrophobic interaction chromatography, whereas the ferredoxins and monooxygenases were expressed as recombinant proteins in E. coli and purified via Ni‑NTA affinity chromatography. For light driven biocatalysis, the conversion of different alkanes to their respective alkanols, the formation of perillyl alcohol from limonene and the epoxidation of aromatic and aliphatic substrates were tested, analyzed and quantified via GC-FID. In addition, P700+ reduction kinetics were established as a screening tool for electron acceptors for PSI.In conclusion, the formation of 1-octanol, 1- hexanol, perillyl alcohol and 1,2-epoxyhexane using the in vitro cascade serves as a proof of concept for PSI driven monooxygenase activity and paves the way for the implementation of CYP153A enzymes in cyanobacteria for in vivo models. Additionally, P700+ reduction kinetics could be utilized to determine the electron transport efficiency between PSI and various potential electron acceptors.

The effect of flavonoids on cyanobacterial motilitiy and their role in symbiotic relationship with land plants

Cyanobacteria have symbiotic relationships with diverse land plants, from bryophytes to ferns, gymnosperms, and angiosperms. One specific example is the symbiosis between Azolla filiculoides and its cyanobiont Nostoc azollae: the cyanobiont is vertically inherited, and both partners co-evolve, which results in full co-dependency. Essential for all cyanobacterial symbioses is the ability to differentiate into motile filaments, hormogonia, upon plant cues. In Azolla, hormogonia is required to colonize new tissue and inheritance; however, the molecular cues underlying the symbiotic transfer are only poorly understood. Salicylic acid (SA) is produced by A. filiculoides and in land plants as a defense response to different pathogenic microbes in the environment. When SA is exogenously applied to A. filiculoides, its cyanobiont's abundance and gene expression are altered. Our preliminary transcriptomic data suggest that SA may control flavonoids, metabolites that in the past have been suggested to influence cyanobacterial motility. We want to test whether SA and flavonoids play a role in cyanobacterial symbiotic relationships. To do that, we investigated the effect of SA and flavonoids produced by A. filiculoides on cyanobacterial symbionts. We induced motility in cyanobacteria using far-red light and tested the ability of the phenolic compounds to modulate the transition into hormogonia stages. The results of the treatments were documented using light microscopy and RNA was extracted for further analyses and then sequenced. We set up a machine-learning-based image analysis pipeline and analysed light microscopy images to quantify differentiation into hormogonia and the amount of vegetative cells. We found that SA and the majority of flavonoids tested enhanced hormogonia transition in a symbiotic cyanobacterium. Now, we are analysing the RNA-Seq data obtained from this experiment to connect differential gene expression to the motility phenotype, hereby focusing on genes known to modulate motility, nitrogen-fixation, and those suggested to be related to the communication between cyanobacteria and their hosts.

A microfluidic droplet-based system for the phototrophic cultivation of cyanobacteria to optimize growth for physiological and analytical data acquisition from bulk to single cell level

Introduction: Microfluidic systems offer the possibility of combining and integrating many process steps in a very small space, enabling highly miniaturized continuous workflows. In combination with miniaturization in the field of quantitative bioanalytic and their integration into microfluidic systems, they are the key to a better understanding of cellular functioning and will also be crucial for further advances in the field of miniaturized and microfluidic from bulk to single cell analysis. Cyanobacteria such as Synechocystis sp. PCC 6803 are photosynthetic microorganisms with considerable biotechnological potential in the bio recovery of commodities, the production of various bioactive compounds and renewable biofuels. And could thus support the transition from the traditional linear economy to a circular economy in the near future. By means of phototrophic growth through oxygenic photosynthesis and the associated ability to produce photobiological hydrogen, the potential of an eco-friendly and renewable future-oriented energy source is also given.Objectives: The microfluidic systems and droplet generation on the pL to nL scale bring bulk cultivation to the level of single cell cultivation for analyzing the heterogeneity of cell populations. Cell proliferation should be trackable online to determine optimal growth conditions and parameters of hydrogen production driven by oxygenic photosynthesis. The microfluidic LOC (Lab-on-Chip) device acts as a photobioreactor in these studies.Materials & methods: Microfluidic chips for droplet generation, incubation and analysis. Light-coupled microfluidic Photobioreactor. Optical online data acquisition and flow cell endpoint determination.Results: Non-invasive optical cell mass determination with sub-pg sensitivity, microfluidic perfusion cultivations for establishing physiological steady-states, and nano- to picoliter batch reactors, enabled the quantification of all physiological parameters. The heterogeneity of the cellular reactivity of single biocatalysts per droplet could be demonstrated by parallelizing the droplet incubation. These results enable the resolution of biocatalysis beyond averages of populations.Conclusion: Microfluidic droplet-based system for the phototrophic cultivation as nano size batch photobioreactors enables the quantification of physiological growth parameter and allows the analysis of the heterogeneity of cell population on from bulk to the single cell level.

Actinospongicola halichondria gen. nov. sp. nov., the first sponge-derived cultivated representative of a new genus within the class Acidimicrobiia

Microbial diversity and evolution

The current, valid taxonomic class Acidimicrobiia includes two orders (Acidimicrobiales, Iamiales), three families (Acidimicrobiaceae, Iamiaceae and Ilumatobacteraceae) and 16 species. Bacteria from the family Acidimicrobiaceae are often described from acid mine drainage or geothermal vents while members of the Iamiaceae and Ilumatobacteraceae are found in neutral or mildly alkaline pH environments, such as marine sediments and invertebrates or the human skin. Recent studies using culture-independent methods have shown a wide distribution of this clade ranging from coastal to deep sea environments as well as various marine invertebrate hosts. Acidimicrobiia isolation has however been notoriously difficult and frequently unsuccessful. In the present study, we report on the isolation of a novel Acidimicrobiia strain Hal371T from the Baltic Sea sponge Halichondria panicea. The strain forms very small, nearly translucent colonies on Baltic Sea agar plates that can easily be overlooked. Based on phylogenetic, genomic, chemotaxonomic, and physiological features, strain Hal317T was described as member of a novel genus of the family Iamiaceae, for which the name Actinospongicola halichondria is proposed. The genome of strain Hal317T exhibited diverse characteristics, ranging from the utilization of diverse carbohydrates, transport of amino acids, osmoprotectants synthesis, defense mechanisms against reactive oxygen species (ROS), chaperones, and toxin antitoxin systems, that may reflect an adaptation to a lifestyle within marine sponges. Strain Hal317T displays further unique genome features that set it apart from other type strains of Iamiaceae, such as a non-phosphorylated L-fucose pathway, a substrate-specific phosphonate pathway, and a glutamate-uptake system. By metabolizing of taurine as well as by uptaking and utilizing of urea and ammonia the isolate might be involved in detoxification processes of sponge-derived metabolic products.

Prof. Dr.

Functional characterisation of the [FeFe] hydrogenase SoHydAB from Shewanella oneidensis

Hydrogenases are metalloenzymes that catalyse the interconversion between two protons and two electrons on one hand and molecular hydrogen (H2) on the other hand. Diverse members of this enzyme family fulfil a wide variety of metabolic functions. The [FeFe] hydrogenase of Shewanella oneidensis presents an intriguing case.This bacterium is well known for its metabolic versatility, allowing it to grow on and reduce an astounding variety of substrates, including heavy metals and highly toxic chemicals. These abilities have made Shewanella a popular choice for the application in microbial fuel cells and wastewater treatment. Due to these properties, the physiology and metabolism of Shewanella has been studied intensively and is well characterised. Biochemically however, the hydrogen metabolism of Shewanella remains understudied.The [FeFe] hydrogenase of Shewanella oneidensis is located in the periplasm, but presumably anchored to the inner membrane. Its activity is completely abolished by the deletion of formate dehydrogenase, but unlike E. coli for example, Shewanella does not appear to form a formate-hydrogen-lyase complex. Furthermore, it has been shown that hydrogenase activity is also fully abolished in the absence of menaquinone.Taken together, the properties of this enzyme and its metabolic context suggest that it either forms a scientifically interesting complex or has a variety of interaction partners, which is likely to lead to further research questions regarding the mechanisms and regulation of these interactions. The first step in elucidating the hydrogen metabolism in Shewanella is a full investigation into the properties of the hydrogenase itself.This poster presents the first characterisation of the [FeFe] hydrogenase SoHydAB utilising an array of chromatographic, electrochemical and spectroscopic methods.

From random mutagenesis to genome editing: Targeting spo0A in Bacillus thuringiensis with CRISPR-Cas9

Bacillus thuringiensis (Bt), a Gram-positive spore former, is a reference biopesticide, largely due to its production of insecticidal 6-endotoxins. However, introducing high quantities of a sporulating organism may prolong its environmental persistence and pose ecological concerns. This study combines random mutagenesis and CRlSPR-Cas9 genome editing to obtain more efficient, eco-friendly Bt strains, focusing on the inactivation of the master sporulation regulator, spo0A.Experimental SetupOur approach comprised two phases:(I) Random mutagenesis generated hyperproductive and oligosporogenic variants of the Bt strain BUPM98, which were screened for toxin production and sporulation.(II) The most promising mutant was chosen for CRISPR-Cas9 genome editing, targeting spo0A to modulate sporulation without undermining toxin output. This strategy pinpoints crucial genomic changes that affect production and sporulation.ResultsFive hyperproductive mutants were identified, three of which displayed markedly Reduced sporulation. Genomic analyses revealed multiple chromosomal rearrangements, frequently mediated by prophages and transposons. Mutant N12 exhibited a 112% surge in toxin production relative to the wild type, with sporulation rates approximating the parental strain (log = 0.04). This outcome correlates with expanded copies of the ylmC gene, likely integral to hyperproduction. In contrast, mutant Nl3 lost several sporulation-related genes, resulting in a reduced sporulation rate (1og2 = -1.34). Guided by these observations, we designed a spo0A- targeting RNA to engineer a non-sporulating strain from N12 while preserving its productivity.ConclusionThese findings highlight the impact of genomic rearrangements and targeted editing on Bt phenotypes. Notably, N12 combines enhanced toxin yield with stable sporulation, making it a promising candidate for sustainable biocontrol solutions. The spo0A guide RNA furtherunderscores CRISPR-Cas9"s potential for refining Bt strains in line with ecological pest management. Overall, merging classical mutagenesis with modem genome editing can tailor Bt to meet specifıc agricultural and environmental needs.KeywordsBacillus thuringiensis, random mutagenesis, genomic rearrangements, sporulation genes, CRISPR-Cas9

Late poster

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