Open Access

The rewetting of formerly drained peatlands can help to counteract climate change through the reduction of CO2 emissions. However, this can lead to resuming CH4 emissions due to changes in the microbiome, favoring CH4-producing archaea. How plants, hydrology and microbiomes interact as ultimate determinants of CH4 dynamics is still poorly understood. Using a mesocosm approach, we studied peat microbiomes, below-ground root biomass and CH4 fluxes with three different water level regimes (stable high, stable low and fluctuating) and four different plant communities (bare peat, Carex rostrata, Juncus inflexus and their mixture) over the course of one growing season. A significant difference in microbiome composition was found between mesocosms with and without plants, while the difference between plant species identity or water regimes was rather weak. A significant difference was also found between the upper and lower peat, with the difference increasing as plants grew. By the end of the growing season, the methanogen relative abundance was higher in the sub-soil layer, as well as in the bare peat and C. rostrata pots, as compared to J. inflexus or mixture pots. This was inversely linked to the larger root area of J. inflexus. The root area also negatively correlated with CH4 fluxes which positively correlated with the relative abundance of methanogens. Despite the absence or low abundance of methanotrophs in many samples, the integration of methanotroph abundance improved the quality of the correlation with CH4 fluxes, and methanogens and methanotrophs together determined CH4 fluxes in a structural equation model. However, water regime showed no significant impact on plant roots and methanogens, and consequently, on CH4 fluxes. This study showed that plant roots determined the microbiome composition and, in particular, the relative abundance of methanogens and methanotrophs, which, in interaction, drove the CH4 fluxes.

During the SARS-CoV-2 pandemic, different viral variants emerged with distinct transmissibility, pathogenicity, and immune evasion features. Although these traits were reflected in the pandemic course, knowledge about immunity against the emerging variants remained limited and lagged behind. Information about early antiviral immunity mainly came from blood or tissue samples of individuals who died from COVID-19, which cannot fully reflect local protective immune responses during acute SARS-CoV-2 infections. This thesis utilized small animals to model COVID-19 in humans and used it afterward to test mRNA vaccine efficacy. The aim was to gain insights into early immune responses after SARS-CoV-2 infection and mRNA-induced protective immunity in various tissues, which is challenging to accomplish in humans. Publication I demonstrates that infection of K18-hACE2 mice with ancestral SARS-CoV- 2, Beta, or Delta VOC results in unique patterns of viral dissemination into the lungs and distinct inflammatory responses within the first 5 days post-infection (DPI). The Beta variant spread to the lungs earlier and caused higher levels of inflammatory cytokines and more significant infiltration of innate immune cells than the ancestral SARS-CoV-2. Adaptive immune responses were detected within 7 DPI and were associated with lower viral burden, suggesting a potential role in clearing the infection. The depletion of B and T cells allowed correlating viral clearance with virus-specific antibody levels and T cell responses. While innate immune responses differed among SARS-CoV-2 variants, adaptive responses developed similarly, suggesting a potential target for pan-variant countermeasures. Publication II indicates that low-dose mixed mRNA vaccines, containing half the amount of each monovalent vaccine, produce similar levels of neutralizing antibodies and virus-specific T cells in the lungs, protecting K18-hACE2 mice from lethal disease. The vaccination of Wistar rats showed that bivalent vaccination can elicit neutralizing antibodies against different variants (e.g., BA1 and BA.5) that are not included in the vaccine formulation. The study suggests that alternative vaccination strategies could generate cross-protective immunity, helping to overcome immune evasion by SARS-CoV-2 variants. Publication III investigates protective immunity against SARS-CoV-2 upon mRNA vaccination in a mouse model with restricted B cell responses, commonly seen in vaccinated individuals with immunodeficiencies. It demonstrates that T cells can compensate for the lack of serum antibodies to prevent sublethal disease in mice. Serum antibody levels correlated with reduced disease severity and mRNA vaccine-induced IgG limited viral replication in the nasal conchae, indicating a potential role in protecting from virus transmission. This thesis provides valuable insights into protective immune responses against infection with various SARS-CoV-2 variants and upon mRNA vaccination and informs about vaccination strategies to overcome immune escape. The numerous experimental studies within this thesis are essential for characterizing emerging viral variants and developing effective vaccines. These findings offer valuable insights that can guide the development of effective countermeasures to combat immune evasion by future SARS-CoV-2 variants.

With 2.56 million deaths worldwide annually, pneumonia is one of the leading causes of death. The most frequent causative pathogens are Streptococcus pneumoniae and influenza A virus. Lately, the interaction between the pathogens, the host, and its microbiome have gained more attention. The microbiome is known to promote the immune response toward pathogens; however, our knowledge on how infections affect the microbiome is still scarce. Here, the impact of colonization and infection with S. pneumoniae and influenza A virus on the structure and function of the respiratory and gastrointestinal microbiomes of mice was investigated. Using a meta-omics approach, we identified specific differences between the bacterial and viral infection. Pneumococcal colonization had minor effects on the taxonomic composition of the respiratory microbiome, while acute infections caused decreased microbial complexity. In contrast, richness was unaffected following H1N1 infection. Within the gastrointestinal microbiome, we found exclusive changes in structure and function, depending on the pathogen. While pneumococcal colonization had no effects on taxonomic composition of the gastrointestinal microbiome, increased abundance of Akkermansiaceae and Spirochaetaceae as well as decreased amounts of Clostridiaceae were exclusively found during invasive S. pneumoniae infection. The presence of Staphylococcaceae was specific for viral pneumonia. Investigation of the intestinal microbiomés functional composition revealed reduced expression of flagellin and rubrerythrin and increased levels of ATPase during pneumococcal infection, while increased amounts of acetyl coenzyme A (acetyl-CoA) acetyltransferase and enoyl-CoA transferase were unique after H1N1 infection. In conclusion, identification of specific taxonomic and functional profiles of the respiratory and gastrointestinal microbiome allowed the discrimination between bacterial and viral pneumonia.

Studying the fates of oil components and their interactions with ecological systems is essential for developing comprehensive management strategies and enhancing restoration following oil spill incidents. The potential expansion of Kazakhstan’s role in the global oil market necessitates the existence of land-specific studies that contribute to the field of bioremediation. In this study, a set of experiments was designed to assess the growth and biodegradation capacities of eight fungal strains sourced from Kazakhstan soil when exposed to the hydrocarbon substrates from which they were initially isolated. The strains were identified as Aspergillus sp. SBUG-M1743, Penicillium javanicum SBUG-M1744, SBUG-M1770, Trichoderma harzianum SBUG-M1750 and Fusarium oxysporum SBUG-1746, SBUG-M1748, SBUG-M1768 and SBUG-M1769 using the internal transcribed spacer (ITS) region. Furthermore, microscopic and macroscopic evaluations agreed with the sequence-based identification. Aspergillus sp. SBUG-M1743 and P. javanicum SBUG-M1744 displayed remarkable biodegradation capabilities in the presence of tetradecane with up to a 9-fold biomass increase in the static cultures. T. harzianum SBUG-M1750 exhibited poor growth, which was a consequence of its low efficiency of tetradecane degradation. Monocarboxylic acids were the main degradation products by SBUG-M1743, SBUG-M1744, SBUG-M1750, and SBUG-M1770 indicating the monoterminal degradation pathway through β-oxidation, while the additional detection of dicarboxylic acid in SBUG-M1768 and SBUG-M1769 cultures was indicative of the fungus’ ability to undertake both monoterminal and diterminal degradation pathways. F. oxysporum SBUG-M1746 and SBUG-M1748 in the presence of cyclohexanone showed a doubling of the biomass with the ability to degrade the substrate almost completely in shake cultures. F. oxysporum SBUG-M1746 was also able to degrade cyclohexane completely and excreted all possible metabolites of the degradation pathway. Understanding the degradation potential of these fungal isolates to different hydrocarbon substrates will help in developing effective bioremediation strategies tailored to local conditions.

For centuries, peatlands have received little attention or recognition due to their perceived insignificance, invisibility, and negative associations. However, they are now receiving increasing attention due to the ecosystem services they provide as carbon and water reservoirs, as well as their rich biodiversity of rare and endangered animals and plants. In the discussion of greenhouse gas (GHG) emissions and their impact on the climate, peatlands are also recognized as being of significant importance. To comprehend the source of these GHG, it is necessary to examine the microbiome of peatlands and the effects of lowering (draining) and raising (rewetting) the water level. Peatlands store large amounts of soil organic carbon (SOC) in form of peat. Dead plant material is preserved in the water-saturated soils and the carbon dioxide (CO2) absorbed from the atmosphere through photosynthesis is stored as peat. However, when the water table in peatlands is lowered, atmospheric oxygen enters the soil, and the soil microbiome changes, leading to the microbial decomposition of SOC and the release of large quantities of CO2. Rewetting can halt aerobic decomposition processes by blocking oxygen. Anaerobic conditions are not conducive to aerobic organisms. Complex plant polymers, such as lignin, can no longer be broken down, preserving the peat body and allowing the peatland to once again serve as a long-term carbon sink. Under these anaerobic conditions, methanogenic archaea produce methane from metabolic products of fermentation processes. Methane is emitted in much smaller quantities, but has about 30 times the warming potential of CO2, making it the second most important GHG after CO2. To gain a better understanding of the processes involved in methanogenesis, it is essential to conduct a detailed investigation of the individual groups of archaea involved. The metabolic pathways of hydrogenotrophic methanogenesis from CO2 and hydrogen, as well as acetoclastic methanogenesis from acetate cleavage, have already been extensively researched and are considered the most significant pathways of methanogenesis. In recent years, additional organisms have been discovered that carry out methylotrophic methanogenesis from methanol and methylated compounds. The methylotrophic order of Methanomassiliicoccales was first described in 2012, but only one pure culture, Methanomassiliicoccus luminyensis, has been isolated. Further research into Methanomassiliicoccales, commonly found in mires, can enhance our understanding of methanogenesis not only in mires but also in other habitats, such as the gastrointestinal tracts (GIT) of animals or water body sediments. This study investigates the peat soil microbiomes, with a focus on methanogenic Archaea of the order Methanomassiliicoccales, at the six study sites of the WETSCAPES project. The sites are located in the three important fen types of Mecklenburg-Western Pomerania (alder forest, percolation fen, and coastal flooded fen), each in drained and rewetted areas. The first article investigates the microbial composition of prokaryotes and eukaryotes in the sites using Illumina MiSeq Amplicon sequencing of 16S rRNA and 18S rRNA genes. The aerobic microorganisms responsible for decomposing plant biomass are present in drained peatlands. In rewetted sites, the relative abundance of these organisms was significantly lower, while other groups, such as fermenting bacteria and methanogenic archaea, occurred in greater numbers. Quantitative polymerase chain reaction (qPCR) was used to determine the number of methanogenic archaea per gram soil. The results indicate that methanogens are at least 10 times more abundant in rewetted peatlands than in drained sites, suggesting increased methanogenesis in rewetted peatlands. These findings were confirmed by GHG measurements. The second article presents an overview of the interactions between water transport, soil chemistry, primary production, peat formation, material conversion and transport, microorganisms, and GHG exchange. The findings are based on state-of-the-art methods in the relevant research areas and combine initial results from all project partners of the WETSCAPES project. The third article focuses on an application-oriented approach and tests different rewetting strategies. The objective of this study was to decrease methane emissions by removing the topsoil and additional planting of peat mosses. The results of the study, which included measurements of GHG emissions and qPCR analyses of methanogenic archaea, showed a significant reduction in methane emissions after the top 30 cm of soil were removed compared to the untreated area. The fourth article deals with the methylotrophic order Methanomassiliicoccales, which belongs to the group of methanogenic archaea. Over a period of 2.5 years, at 12 time points, at three soil depths, 16S rRNA gene sequencing was used to produce a seasonal and spatial analysis of the Methanomassiliicoccales phylotypes present. The results revealed a distribution of the phylotypes based on soil depth, redox potential, and season, which were related to their physiological characteristics. In the fifth article, the 1.52 Mbp genome of "Ca. Methanogranum gryphiswaldense U3.2.1", a Methanomassiliicoccales strain, enriched to > 80% from peat soil of the rewetted percolation fen, was presented. The sixth article presented the microscopic, physiological, and genomic studies of Ca. Methanogranum gryphiswaldense U3.2.1. This hydrogen-dependent, methylotrophic strain from the family Methanomethylophilaceae, uses methanol and trimethylamine as electron acceptors. Despite various attempts, a pure culture has not yet been achieved. The study demonstrates the impact of rewetting on the soil microbiome, revealing its complexity. The development of the microbiome was influenced by various biotic and abiotic factors, depending on the location and water level. The abundance of methanogenic archaea was significantly higher in the rewetted sites compared to the drained ones. The Methanomassiliicoccales were found to be a significant contributor to the methanogenic archaea in the investigated sites. It remains to be examinated whether they also significantly contribute to methane formation. Enrichment cultures and genome analyses were used to investigate the substrate spectrum of Ca. Methanogranum gryphiswaldense U3.2.1 obtained from peat soil. The extraction of a pure culture from peat soils has not yet been successful and therefore remains a goal of future investigations.