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

Prof.

Promoting autotrophic denitrification in shallow porous aquifers for drinking water production

Environmental microbiology & ecology

Nitrate pollution in groundwater is a global challenge since groundwater serves as a vital drinking water source across Europe. Avoiding a nitrate overload requires sustainable management schemes for shallow aquifers with high groundwater recharge. However, their attenuation potential against pollutants like nitrate is often limited. High nitrate levels persist in many aquifers even long after reducing nitrogen inputs. Especially for otherwise oligotrophic shallow groundwater systems, innovative and cost-effective technologies to eliminate nitrate loading and to ensure a sustainable supply of drinking and utility water are urgently needed to meet rising water demands.In this project we will implement a novel approach for nitrate removal from shallow groundwater. Via the injection of reduced gaseous electron donors (H₂ & CH₄), we aim to stimulate autotrophic denitrification directly upstream of burdened drinking water wells. This has the advantage of avoiding an enrichment of heterotrophic, potentially pathogenic microorganisms.In lab scale microcosms, we have simulated a range of in-situ conditions and aquifer materials. We identified temperature as a key driver for denitrification rates. Enrichment cultures of autotrophic denitrifying microorganisms for a potential augmentation and stimulation of denitrification inside natural aquifers were successfully introduced to a sediment matrix in a bioreactor. We inoculated non-denitrifying aquifer material with enriched stable denitrifying communities and successfully reestablished denitrification in the presence of hydrogen and methane. In a currently running mesoscale pilot plant experiment we are testing the principles of gas injection into porous media and monitor the stimulation effects over time. In a final stage, we hope to successfully implement our approach at the field scale to eliminate nitrate in an actual drinking water production system.We aim to offer proof that injecting H₂/CH₄ can effectively eliminate nitrate from groundwater aquifers without adversely impacting water quality or hydraulic characteristics through secondary reactions, like mineral dissolution and precipitation. Harnessing the intrinsic potential of autotrophic microorganisms native to groundwater systems and using gasses that can be produced with green biotechnology offers a new and sustainable solution to a notorious problem.

Poster

Prof. Dr.

Resistance properties among different Clostridioides difficile isolates

The rod-shaped, motile and obligate anaerobic bacterium Clostridioides difficile occurs ubiquitously in the environment and colonizes the gastrointestinal tract of mammals. As a leading causative agent of antibiotic-associated diarrhea worldwide, it is particularly relevant in clinical settings. The pathogenicity of C. difficile is based on the formation and secretion of toxins and the associated damage to the intestinal epithelial tissue. If the C. difficile infection (CDI) progresses severely, the consequences for the patient can be fatal. Treating CDI is challenging due to C. difficile's ability to form metabolic dormant endospores, which leads to the risk of reinfection.Since the first description in the 1930s, numerous isolates have been characterized. Common protocols recommend heat or ethanol shock during sample preparation, using the endospore's resistance for isolating purposes. It became apparent that both, the vegetative cells and the corresponding endospores of several isolates react differently to certain treatments. As a result, the isolation conditions used to monitor C. difficile populations might not reflect the actual situation. To address this issue, a panel of C. difficile isolates were tested regarding their resistance properties over different exposure times applying a plating procedure. Here, defined concentrations of vegetative cells as well as endospores were used to determine classical characterization parameters based on the inactivation kinetics. This knowledge provides information to improve isolation and enrichment strategies to obtain a better understanding of the environmental distribution of C. difficile populations.

Influence of oxygen supply on bacterial degradation of various azo dyes by Rhodoccocus opacus 1CP

Azo dyes are a primary group of synthetic dyes widely used for textiles, food technology, and paper printing. These compounds contain one or more azo bonds linking aromatic rings. Some dyes and their aromatic biotransformation products are toxic or carcinogenic, which makes them significant environmental pollutants. Azo dyes can be degraded by microorganisms under anaerobic, facultative aerobic, or aerobic conditions, depending on the type of organism used. Anaerobic degradation involves nonspecific enzymatic processes, whereby resulting amine compounds cannot be further broken down under these conditions and may be toxic. Under aerobic conditions, these amine compounds can be further degraded. It has also been shown that oxygen can inhibit dye decolorization, as evidenced by comparing static and shaking culture conditions. Additionally, the primary organic carbon source present can influence the degradation process under both anaerobic and aerobic conditions.However, the effects of varying oxygen supply levels remain not fully understood, particularly for the organism Rhodococcus opacus 1CP. The study will focus on investigating the influence of oxygen supply on biodegradation efficiency. Oxygen transfer rate (OTR) measurements in 96-well plates allow for varying oxygen availability for bacterial cultures depending on the filling volume of each well. This high-throughput screening method enables simultaneous investigation of numerous different settings, combining azo dye decolorization measurements with mass spectrometric analysis of degradation products to examine the impact of dissolved oxygen on the complete biomineralization of various azo dyes.Experiments confirmed the theory that rapid decolorization of the azo dyes methyl red and brilliant black occurs at lower OTRs due to reduced oxygen availability by the organism R. opacus 1CP. Additionally, a significant difference in the OTR profile during the cultivation of the organism was observed.The data provided deeper insight into these processes, enabling targeted adaptations for cultivation settings that could lead to more efficient azo dye decolorization and by-product degradation through optimized oxygen supply.

Forest tree holes as models to study the island biogeography of microorganisms

Water-filled tree holes are naturally formed semi-enclosed cavities in which environmental conditions such as water availability strongly fluctuate. These small aquatic ecosystems feature island-like characteristics that render them suitable models to test island biogeography theory and to study the mechanisms of microbial community assembly.Our study was conducted within the German Biodiversity Exploratories framework and is one of the first targeting both bacterial and fungal communities present in tree holes. Microbial biomass collected from 55 water-filled tree holes (predominately Fagus sylvatica) in three different geographic regions were analyzed by Illumina high-throughput amplicon sequencing (bacteria: V3 16S rRNA gene, fungi: ITS2 rRNA gene region). Community composition was used to elucidate the factors shaping biogeographic patterns and to examine the extent to which tree holes represent microbial islands.Tree holes were found to harbour complex microbial communities. We observed bacterial species known to be associated with freshwater and sediment (Limnohabitans, Flavobacterium, Polynucleobacter), soil (Pseudomonas) and plant-associated (degrading) (Duganella, Paludibacter) environments, as well as phytopathogenic (Fusarium), insect-associated (Zoophthora), and aquatic fungi (Alatospora, Flagellospora, Gorgomyces) including yeasts (Camptobasidium). Overall, we observed a limited overlap of bacterial or fungal taxa between individual tree holes. In contrast to previous reports, species richness of the bacterial communities decreased with increasing tree hole volume. However, we found evidence that deterministic processes had little overall influence on microbial community assembly, whereas stochastic processes were predominant in these systems. Especially dispersal limitation was important, which had a greater impact on bacteria than on fungi. Since community assembly was largely mediated by stochastic processes, our results support the conclusion that tree holes act as islands for microbial communities.

Miss

Targeted ecological effects of phage cocktails against a pathogen of foals in the soil of horse breeding farms

Virulent Rhodococcus equi is a globally recognised pathogen of young foals, best known for causing aggressive pyogranulomatous bronchopneumonia. The lack of a vaccine, emergence of multi-drug resistance and the soil-animal lifestyle of R. equi necessitates novel approaches to disease control. The specificity of bacteriophages (phages) offers a promising opportunity to manage R. equi burdens in soil. The aim of this study was to formulate phage cocktails (combinations of phage) and assess their capacity for R. equi burden control in soil. This study involved in vitro laboratory and soil intervention experimental design. Five phage candidates isolated from fecal and soil samples from Thoroughbred farms were screened against a collection of virulent R. equi isolates. Collectively, the phages showed lytic activity against 20/22 historical (1991-2004) and 17/21 contemporary (2021-2023) environmental and clinical R. equi isolates. Two cocktails were trialled against mixed R. equi populations and were able to limit bacterial growth to within 1.5 log CFU/g of the starting concentration in sterile soil and actively reduce total R. equi populations by 1 log CFU/g in natural soil. Additional parameters of successful application include phage persistence and analysis of the effect of phage on the surrounding soil microbiota. This work demonstrates the targeted and robust nature of phages as bactericidal agents in the soil environment.

Prof., Dr.

Deciphering the cabanillasin biosynthesis pathway using limited proteolysis coupled with mass spectrometry (LiP-MS)

Cabanillasins are rare polyguanidine natural products from entomopathogenic Xenorhabdus bacteria with good antifungal bioactivity.Although we were able to identify building blocks and intermediates in the biosynthesis of these unusual compounds, we were not able to identify the actual biosynthetic gene cluster (BGC) nor the genes or enzymes involved in their biosynthesis. To address this knowledge gap, we wanted to apply limited proteolysis coupled with mass spectrometry (LiP-MS), a proteomics method developed by the group of Paola Picotti in 2017. We adjusted it to study the Xenorhabdus doucetiae proteome, and to find the proteins involved in the cabanillasin biosynthesis pathway. Briefly, the proteome of X. doucetiae was studied in the absence or presence of building blocks and precursor already identified in the cabanillasin biosynthesis by digestion using proteinase K (PK), followed by trypsin digestion.The difference in the overall protein structures binding to the building blocks and intermediates led to different cleavage sites for PK and trypsin, resulting in different peptides (in the absence or presence of the building blocks), and enabling the identification of candidate proteins probably involved in the cabanillasin biosynthesis pathway.We also tested LiP-MS to identify candidate targets of bioactive natural products showing antibiotic activity since the advantage of this method is that it does not require the chemical modification of the original natural product and is therefore also suitable for very low molecular weight natural products.

Investigations on bile acid catabolism in Sphingobium sp. strain Chol11 suggests a Rieske monooxygenase-catalyzed hydroxylation that occurs prior to the degradation of the carboxylic side chain

Bile acids are steroids produced by vertebrates in their digestive system, and after excretion, they can be used as carbon and energy sources by environmental bacteria. Bile acids have a hydroxylated steroid skeleton with a C5 carboxylic side chain attached to C17. Two pathway variants, named Δ1,4 and Δ4,6, are known for bile acid catabolism under aerobic conditions. While the Δ1,4-variant is common in pseudomonads and Actinobacteria, the Δ4,6-variant seems unique to the Sphingomonadaceae family.Proteomic analysis of Sphingobium sp. strain Chol11 grown with bile acids revealed a gene cluster specifically upregulated during side-chain degradation, which is conserved in this family. It contains genes for a CoA ligase (sclA), an acyl-CoA-dehydrogenase (scd4AB), as well as genes for an amidase and a Rieske monooxygenase (RMO). Notably, this cluster lacks genes for the stepwise side-chain degradation described in bacteria using the Δ1,4 variant. Additionally, Chol11 Δscd4A cannot grow with bile acids and accumulates hydroxylated dead-end metabolites with a C5 side-chain. These instances suggest that side-chain degradation in strain Chol11 involves a hydroxylation catalyzed by the RMO.The potential role of the RMO was investigated through gene deletion and complementation experiments. Gene deletion attempts led to the removal of a 64 kb genomic region, encompassing a substantial fraction of the side-chain degradation cluster containing the genes coding for the RMO, the amidase, and the acyl-CoA dehydrogenase Scd4AB, plus a downstream genomic region. Complementation of this mutant with the genes coding for RMO only, as well as with the amidase and RMO genes together, resulted in the production of hydroxylated metabolites containing an intact side chain. These metabolites resembled the dead-end metabolite production by strain Chol11 Δscd4A. These results indicate that RMO catalyzes the hydroxylation of intermediates in the Δscd4A mutant and suggest that these hydroxylated compounds might be the substrate for Scd4AB before the side-chain degradation.This study provides further evidence that bile acid side-chain degradation in Sphingomonadaceae proceeds via an uncharacterized pathway involving an RMO-catalyzed hydroxylation. Current efforts are focused on complementing strain Chol11 m25 with different gene combinations comprising scd4AB, RMO, and amidase genes in order to elucidate the putative reaction sequence catalyzed by the respective enzymes.

Identification of microbial keystones in complex soil ecosystems and determination of their activity and niche preferences

Determining the functional role of individual types of microorganisms and their specific relevance in the environment remains very challenging, especially in highly complex environments such as soils. One promising approach is to try to detect and characterize so-called keystone taxa, which are critical to the stability and functioning of the entire communities. We used co-occurrence networks from high-throughput sequencing data to elucidate significant associations, and to thereby infer microbial sequence types that might be of particular importance to the overall microbial communities, hence representing keystones. This approach was applied to bacterial and fungal communities in 300 different soils of the German Biodiversity Exploratories. The sites encompass grassland and forest ecosystems under different land use regimes, which were sampled over a period of 11 years. 16S rRNA and the internal transcribed spacer (ITS2) were used as markers for bacteria and fungi, respectively. Keystones were identified by a ranking procedure based on the susceptible-infected-recovered model after perturbation of the original networks. Keystone taxa were specific to distinct soil environments within different geographic regions and remained rather stable in their habitat over time. We hypothesized that keystone taxa must be more active than co-occurring microorganisms to exert their ecological influence over longer time periods. Accordingly, active bacterial taxa were identified using both 16S rRNA/ 16S rDNA ratios and also by determining bacterial growth rate from the peak-to-trough ratio of genomic sequence coverage. Notably, we found a significant overlap between keystone and active species, corroborating the network analysis approach. Using the molecular signatures of keystone taxa, their distinct ecological niche preferences were analysed by modelling abundances against environmental parameters, and metagenome assembled genomes (MAGs) from bacterial keystones provided further insights into their functional potential. Our results thus pave the way towards dissecting the ecological roles of individual microbial taxa in highly complex environments.

Bacterial bile acid degradation as a parameter to evaluate effects of a fungicide on selected bacterial taxa both in soil microcosms and in laboratory cultures

Plant protection products (PPPs) have effects on non-target organisms including prokaryotes. In soil this can impair the transformation of organic and inorganic matter. For regulatory purposes, the influence of PPPs on nitrification and respiration is measured but this does not provide information about the affected taxa. For this, a substrate that can only be degraded by selected bacteria, which do occur ubiquitously in soil, would be desirable. Bile acids could be a suitable substrate because they are excreted by all vertebrates and can be used as a carbon and energy source by only a few defined bacterial taxa frequently found in soil. These taxa mainly comprise the genera Pseudomonas, Comamonas and Rhodococcus all of which are also known as degraders of synthetic compounds.It was investigated whether the microbial community in a reference soil (01A) without addition of other nutrients can degrade different bile acids. Chromatographic analyses coupled to mass spectrometry showed that degradation of bile acids instantly started in soil microcosms. Determination of colony forming units revealed an enrichment of bile-acid degrading bacteria. Amplicon sequencing of 16S rRNA genes showed that bacteria of the genera Pseudomonas and Comamonas accumulated in the soil microcosms with cholate and ursodeoxycholate, respectively, which was confirmed by quantitative PCR and isolation of abundant bacteria. Such soil microcosms were then used to analyze the effect of the fungicides Propamocarb and Thiram on bile acid degradation. While Propamocarb had no effect, Thiram delayed degradation of both bile acids. Molecular analysis showed that Thiram reduced the proportion of Pseudomonas in cholate-containing microcosms while the proportion of Comomonas increased. Laboratory cultures with two different isolated strains of each genus showed that Thiram inhibited the degradation of cholate and also of succinate much more in the Pseudomonas than in the Comamonas strains.These results showed that bile acids are a suitable substrate for addressing the metabolic activity of defined bacterial taxa in soils because the bile acids, which only slightly differ in the number and configuration of hydroxyl groups, were utilized by different taxa. This selective enrichment could be used to analyze the effect of a PPP because it consistently showed stronger inhibition of cholate degradation for Pseudomonas than for Comamonas both in soil communities and in laboratory cultures.

Diversity and distribution of genomic potential for sulfoquinovose degradation across gut and soil microbiomes

IntroductionSulfoquinovose is a sulfonated derivate of glucose and one of the most abundant organosulfur compounds in the biosphere. As headgroup of the sulfonolipid sulfoquinovosyl diacylglycerol (SQDG) it is an integral component of thylakoids of photosynthetic organism. So far at least five bacterial degradation mechanisms have been described, which are all initialized by the cleavage of sulfoquinovose from SQDG. Sulfoquinovose is then degraded via transaldolase or transketolase-driven sulfoglycolytic variants of the pentosephosphate, the Embden-Meyerhof-Parnas or the Entner-Doudorff pathway, or an oxygen dependent mono- or dioxygenase driven pathway. Although these pathways differ in their degradation products and contribution to sulfur and carbon cycles, their distribution across environmental and host-associated microbiomes is not fully explored.GoalsHere, we developed a bioinformatic pipeline for the automated generation of hidden Markov models (HMM). We applied this tool for genome-based analysis of microbial SQDG degradation to study the diversity and distribution of SQDG catabolism across soil habitats and in the mouse gut.Material & MethodsThe selection of functionally equivalent reference sequences for the creation of HMMs is a time-consuming task, which was accelerated by the development of an automatized HMM generation pipeline. HMMs are automatically generated based on the combined sequence similarity, genomic vicinity and phylogenetic information from the sequences of a single gene cluster. This tool enabled the automated generation of HMMs that differentiate between the enzymes of glucose and sulfoquinovose catabolism.ResultsThe reliability of the HMMs was verified for several sulfur metabolism related gene clusters, including those of the sulfoquinovose catabolism, in a cross-validation procedure. When applied to large collections of metagenome-assembled genomes from soil and gut microbiomes, mono- and dioxygenase driven aerobic SQDG degradation pathways and the sulfo-Entner-Doudoroff pathway were primarily detected by the generated HMMs. In contrast, the sulfo-Embden-Meyerhof-Parnas and transaldolase driven pathways were more abundant in gut microbiomes, revealing an ecosystem dependent pattern in SQDG catabolism diversity.SummaryDepending on the ecosystem, the genetically predominant sulfoquinovose catabolism differs, which probably affects the availability of sulfur and organosulfur compounds.

Buried Secrets – A journey into subsurface habitats

Beneath our feet lies a world full of mystery—a realm where secrets of life await discovery. Inour investigation of subsurface habitats, including groundwater and seepage water from theHainich CZE (Critical Zone Exploratory) and a cave in Baden-Württemberg, we initiated a questto cultivate the hidden diversity of subsurface habitats and the untapped microbial life theyharbor. The main aim of the investigation: planctomycetes, with their complex cell structure,multiple genes of unknown function and partly mysterious lifestyles.Equipped with cultivation techniques that employed both oxic and anoxic approaches, weproceeded to investigate these enigmatic environments. Each habitat with varying nutrient-,oxygen-, and pH- conditions but with the one common factor of being completely shroudedin darkness.As the cultivation of these samples proceeded, a sense of anticipation arose from thedarkness. Who are these microbial players, and what secrets might they hold? And then on arandom Friday the breakthrough was achieved – The first groundwater Planctomycete wasisolated from an enormous number of plates. And there was more to come, more toinvestigate and more to be fascinated by. Several isolates from the five phyla ofActinomycetota, Bacilli, Bacteroidota, Planctomycetota, and Pseudomonadota were obtained,including novel species that had never seen the light of the day.An overview of these isolates and novel species for all investigated subsurface habitats and acharacterisation of all isolated planctomycetal species will be presented to uncover the buriedsecrets of subsurface habitats.

Intercropping wheat and aromatic crops: Impacts on the root associated microbiome

The root microbiome plays a crucial role in plant health, growth, and yield promotion. Under draught stress, the important roles played by root associated bacteria and arbuscular mycorrhiza (AMF) are more pronounce. Mixed cropping which involves the cultivation of different crops enables the efficient utilization of nutrient by combining deep rooters and shallow rooting plants during a production time period. This enables increased plant diversity and hence diversity of root exudates. The composition of root associated microbiome is controlled by the production of root exudates. The extent of the effects of mixed cropping/intercropping involving wheat and medicinal aromatic plants on the root microbiome is not well studied.In the TRIO project intercropping of wheat-aromatic crops (wheat-coriander, wheat-caraway) and monocrops (wheat, caraway, and coriander) will be investigated to determine the effects of intercropping systems on the root associated microbiome. The project involves two experimental fields under ecological and two experimental fields under conventional agricultural management system in Hesse. The field trials in the four experimental fields will be carried out under randomized complete block experimental design. Each field has 20 plots with 12 plots under monocrops of wheat, caraway, and coriander. For mixed crops eight plots are studied, four plots with wheat-coriander and four plots with wheat-caraway combinations. Annual and perennial crop production will be studied. The microbial diversity and community composition in bulk soil (at sowing and flowering of herbs) and rhizosphere and roots samples at flowering of wheat and herbs will be compared.Our first hypothesis is that mixed cultures have a positive effect on the bacterial diversity and affect nutrient uptake. To answer this hypothesis, the bacterial communities will be analysed by 16S rRNA gene amplicon next generation sequencing. We also hypothesize that crop diversity in mixed crops positively affect AMF colonization benefitting perennial as compared to annual crops. To answer this, fluorescence microscopy of plant in monocrops and mixed will be carried out to determine the colonization abundances of AMF. Quantification of root colonizing AMF using qPCR and diversity analysis using amplicon sequencing will be carried out in parallel.

Microbial abundance and community composition in topsoil profiles of differently cropped and fertilized arable soils

Soil microbial communities are crucial for ecological processes such as organic matter turnover and nitrogen cycling in agricultural soils. In the LOEWE project Green Dairy, we investigated the effects of two different cow manures from high- and low productivity feeding and crop rotatio on the soil microbiome in organic farming systems. Bulk soil samples of 0-10 and 10-30 cm depth collected in spring 2024 from eight arable plots were comparatively analyzed. Alfalfa (one and two year old), spelt, maize, potatoes, oats, rye, wheat and field beans were grown on the plots. Using quantitative PCR (qPCR), we determined the total abundance of bacteria, archaea and fungi as well as ammonium oxidizing bacteria and archaea (detected by amoA genes). In addition, we analyze the diversity and phylogenetic composition of bacterial and archaeal communities by 16S rRNA gene amplicon Illumina sequencing. Current results showed that the abundance of bacteria, archaea and amoA genes in bulk soil was effected by crops. Different manure types only affected the abundance of bacterial 16S rRNA and archaeal amoA genes. In general no significant differences were obtained among the two studied top soil layers. These findings underline the influence of crop rotation on the soil microbiome and thus on soil fertility and plant performance and provide important information for the sustainable management of agricultural land.

Insights into amino acid utilization and regulation in Galdieria sulphuraria

The extremophilic microalga Galdieria sulphuraria thrives in nutrient-limited, fluctuating environments, displaying remarkable metabolic adaptability. We investigated this adaptability through intra- and extracellular metabolomics using LC-MS, complemented by heavy isotope tracers to track amino acid and carbon fate. Our study examined how G. sulphuraria modulates amino acid metabolism in response to environmental amino acid and carbon availability, assessing uptake rates, inhibitory interactions, and metabolic pathway shifts. By following labeled amino acids and carbon, we mapped key metabolic adjustments that support resilience in challenging conditions. These findings provide new insights into G. sulphuraria"s regulatory mechanisms and nutrient utilization strategies, underscoring its potential in biotechnological applications for environments with limited resources.

Environmental microbiology & ecology 1

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