7th joint conference "Joint Microbiology & Infection Conference 2024" of the DGHM and VAAM

Chair

Prof. Dr.

PD. Dr.

Predatory and parasitic microeukaryotes in wastewater treatment plants - diversity &amp; function

Environmental Microbiology & Processes

Microeukaryotes, in particular protists, have been relatively overlooked in wastewater treatment studies due to methodological biases favoring prokaryotes. Applying metatranscriptomics has enabled us to address these biases, and in this presentation, I will provide a summary of four recent studies examining the entire wastewater microbiome, encompassing prokaryotes, fungi, protists, and microscopic metazoa (Heck et al. 2023, Freudenthal et al. 2022, Pohl et al. 2021, Solbach et al. 2021).Our primary focus has been on exploring biotic interactions within wastewater, leading to several noteworthy findings: 1. Microeukaryotic predators shape the prokaryotic community composition and thus are integral for well-performed wastewater treatment. 2. Gut parasites among microeukaryotes exhibit heightened activity in wastewater but undergo effective reduction during denitrification, likely influenced by microbial predation. 3. The so-far overlooked, but most abundant wastewater protist worldwide, Rhogostoma minus, hosts Legionellales and potentially functions as a vector for Legionnaire's disease in humans. These findings underscore the significance of investigating microbial eukaryotes in wastewater, now made feasible with advanced tools.

Oral Presentation

Professor

Reduction of pathogens and (antibiotic-resistant) bacteria in advanced sewage treatment by membrane bioreactor systems and retention soil filters

Sewage treatment plants (STPs) are important punctual sources of antimicrobial resistance spread, including antibiotic-resistant bacteria (ARB) [1]. Thus, an advanced wastewater treatment gained importance in order to decrease microbial load from the discharge of STPs, which enter freshwater systems.In this study, two STPs with membrane bioreactor systems (MBRs) as advanced technologies in the activated sludge process were examined for their reduction performance for hygienically relevant pathogens and ARB. A retention soil filter (RSF) was tested at another STP as an additional treatment stage after conventional wastewater treatment. The reduction of ESBL (extended spectrum β-lactamase)-producing bacteria and VRE (vancomycin-resistant enterococci) as well as accompanying pathogens are in focus of this study. The MBRs and the RSF are running each on municipal STPs in Germany, operated by the Erftverband. Somatic coliphages, Clostridium (perfringens), E. coli, intestinal enterococci, target species of ESBL-producing bacteria (E. coli, Klebsiella spp., Enterococcus spp., Citrobacter spp., Pseudomonas (aeruginosa), Acinetobacter (baumannii)) and VRE were analyzed by cultural detection.The purification performances of the tested species in the STPs with MBRs showed a reduction of 1-2 log10 units of colony-forming units (CFU/100 mL) higher than STPs with conventional wastewater treatment (Fig. 1 A). The RSF showed a reduction performance for e.g. ESBL-E. coli and E. coli of 1.2 and 3.4 log10 units (CFU/100 mL; mean values). ARB could only be detected in rare cases (Fig. 1 B). In total, both RSF and MBRs showed high potential in reduction of ARB and other hygienically relevant parameter. Thus, the treatment techniques offer benefits in reducing pathogens and preventing the dissemination of resistance into the environment.Sib, E., Lenz-Plet, F., Barabasch, V., Klanke, U., Savin, M., Hembach, N., Schallenberg, A., Kehl, K., Albert, C. and Gajdiss, C. Bacteria isolated from hospital, municipal and slaughterhouse wastewaters show characteristic, different resistance profiles. Science of The Total Environment, 2020, 746, 140894, doi:10.1016/j.scitotenv.2020.140894.

Fertilization with microalgae-biomass from wastewater treatment ponds can transmit fecal indicator bacteria into the agricultural system and has specific effects on the root microbiome composition of wheat plants

High rate algal ponds (HRAPs) are effective alternative wastewater treatment systems for sustainable nutrient recycling. The nutrient rich microalgae biomass (MB) can be used as efficient agricultural biofertilizer to replace mineral fertilizers.Complex effects are expected for the plant microbiome due to MB absorbed organic compounds, pollutants and attached bacteria including wastewater derived pathogens and antibiotic resistant bacteria. We studied the release of potential pathogenic, antimicrobial resistant and biocide tolerant bacteria and effects on community structure of the plant microbiome after fertilization with dried MB.Wheat plants in a greenhouse pot experiment were fertilized either with dried MB or mineral fertilizer supplying 60, 120, and 180 kg ha-1 N-equivalents. The root microbiome of fertilized plants was compared to that of non-fertilized plants.Cultivation dependent methods detected E. coli, ESBL-E. coli, enterococci and biocide tolerant bacteria. Total DNA was extracted from rhizospheres and roots for 16S rRNA gene amplicon sequencing and quantification of bacteria, fungi, nitrogen cycling and antibiotic resistant genes (ARG). Compared to control plants, both fertilizers supported plant growth and yield (Mückschel et al. 2023). E. coli, ESBL-E. coli and enterococci were only cultured from MB and roots of MB-fertilized plants but not from potting soil and wheat seedlings used for the experiment. Biocide-tolerant Pseudomonas were cultured from all samples, while biocide tolerant E. coli, Klebsiella and Enterobacter were only cultured after MB application.While the abundance of root colonizing bacteria, fungi and N cycling genes showed no significant difference after MB and mineral fertilizer application, clear differences were obtained for the diversities and composition of the rhizosphere and root microbiome. Plant colonization with MB derived bacteria was thereby indicated. In addition, higher abundance of ARGs was obtained after MB application.Our data showed that fertilizations with HRAP derived MB has complex effects on agricultural ecosystems and still requires more detailed multidisciplinary research.

A molecular approach on understanding seasonal effects on methane-related microbial processes in permafrost-affected soils from West Greenland

The warming Arctic is subjecting permafrost soils, which cover approximately 15 % of the northern land area, to drastically changing conditions. Thawing permafrost enables formerly inactive microbes to produce or oxidize methane (CH4), a potent greenhouse gas. We present first data on seasonal observations of the microbial soil metagenome along two tundra soil moisture gradients on Disko Island, West Greenland.Initial studies have been performed at a time of maximum active layer thickness in September 2022 and during snowmelt in June/July 2023. We show through metabarcoding (16S rRNA gene), that known methane oxidizers (methanotrophs) ubiquitously occurred at low relative levels throughout the whole soil community in September 2022, generally accounting for &lt;1.5 % of all ASVs. During snowmelt in June/July 2023, methanotrophs appeared absent from all surface samples which were at dry to intermediately dry plots. They were mostly detectable below the active thawed layer (below ~10 cm). Methane producing archaea (methanogens) were predominantly present at the wet sites and at deeper depths – as it was expected based on anaerobic incubation experiments with the same samples. Similar to methanotrophs, they were much more prevalent at the thaw maximum in September than during snowmelt in June/July, adding up to almost ten times the amount of detected methanogens in June/July in their relative abundance.Data from two additional field trips at times of the maximum active layer thickness (September 2023) and completely frozen and snow-covered soil (April 2024) will enable further insights into the seasonal variation of methane-related microbial communities. This data set will also be complemented by whole-genome metagenomics, adding information on seasonal changes in methane production and oxidation pathways. A final qPCR approach will add quantitative information to the metabarcoding-derived data. This way, we will be able to help understand the microbial functioning underlying CH4-oxidation and production in different seasons, which in turn will be important for constraining the Arctic CH4 budget, specifically during the shoulder seasons.

Unveiling Microbial Adaptations to High CO2 Environments: Metagenomic Insights from the Eger Rift (Czech Republic)

Introduction High CO2 environments, such as the Hartoušov mofette system in Central Europe&quot;s Eger Rift, harbor diverse microbial communities critical for understanding carbon cycling and extreme condition adaptation1. Metagenomic analysis has emerged as a powerful tool for combing the genetic makeup and metabolic capabilities of these microbial communities. Goals In this study, our objective was to characterize the microbial composition in the CO2-rich environment at the Eger Rift site. Additionally, we analyzed their metabolic potential, focusing on carboxylation reactions involved in fixing CO2 in cellular material. Methods Drill core and water samples were collected from various depths within a drill hole at the Eger Rift site and subjected to metagenomic sequencing. Taxonomic profiles were determined using Kaiju and Kraken2. Metagenome-Assembled Genomes (MAGs) were generated for single organisms, and gene prediction (Prodigal) and functional annotation (eggNOG-mapper) were performed. Results Metagenomic data analysis unveiled a diverse taxonomic landscape of bacteria and archaea in the high CO2 environment of the Eger Rift. Predominant phyla in drill core samples included Pseudomonadota and Cyanobacteria, while Firmicutes, Pseudomonadota, and Bacteroidetes dominated water samples. Two high-quality MAGs were retrieved from the metagenomic samples. One MAG could be identified as the genome of an archaeon, Methanobacterium paludis, which shows an estimated average nucleotide identity of 89.84% and 93.62% with the published reference organisms. The other MAG was affiliated with the genus Proteiniphilum. Within these MAGs, the presence of diverse carbon fixation pathways, including key enzymatic steps and metabolic routes, was identified. Summary Our study extensively explored taxonomic diversity, functional potential, and genetic adaptations in the CO2-rich Hartoušov mofette system. These insights into carbon fixation pathways hold promise for CO2-based biotechnological advancements. References 1. Liu, Q. et al. Microbial Signatures in Deep CO2-Saturated Miocene Sediments of the Active Hartoušov Mofette System (NW Czech Republic). Front. Microbiol. 11, (2020)

Discovering microbial communities within the deep biosphere of arid and humid ecosystems.

The deep biosphere is one of the most extensive and enduring habitats, comprising a significant fraction of Earth"s total biomass. Microbes inhabiting the pore space of sedimentary and igneous rocks in the subsurface encounter various challenges, including anoxic, xeric, and oligotrophic conditions. The initially organic-free igneous systems are nutrient-limited. Thus, they depend on biomass input from the surface through fluid transport along tectonic fractures. Little is known about processes in the deep subsurface of arid landscapes, where surface water input is extremely limited.Our study provides insights into the deep biosphere of arid (Pan de Azúcar) and humid (Nahuelbuta) ecosystems along the coast of Chile. We analyzed microbial communities from up to 55 m deep subsurface granite rocks using 16S rRNA gene amplicon and shotgun metagenomics sequencing. By integrating DNA-based taxonomic and fluorescein diacetate hydrolytic activity (FDA) data, we identified a diverse deep biosphere microbial community beneath both the humid and arid surface conditions showing a potentially higher activity level compared to shallow desert soil communities. This community was characterised mainly by ubiquitous heterotrophic bacteria assigned to Pseudarthrobacter, Janthinobacterium, and Pseudomonas, regardless of surface climate. However, rare taxa affiliated with common chemolithoautotrophs, e.g., Thiobacillus, Sulfuriverula, and Sulfuricurvum, were only observed in the arid subsurface of Pan de Azúcar. This indicates an increased adaption to nutrient-limiting conditions in the arid desert deep biosphere. Functional analyses revealed sulfur and carbon monoxide as the most likely alternative electron donors, while no potential evidence for hydrogenotrophy or iron cycling was detected in both climates. We discovered a diverse and potentially active deep biosphere inhabiting granitic rocks, a system partially influenced by climate conditions on Earth's surface. This contributes to the knowledge about energy acquisition in deep biosphere ecosystems and its interconnection with surface conditions.

Oral Session
Environmental Microbiology & Processes 1

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Oral SessionEnvironmental Microbiology & Processes 1

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Oral Session
Environmental Microbiology & Processes 1