Soil Multi-Meta-Omics: Unraveling microbial regulation at three post-genomic levels
Doctoral thesis
Date of Examination:2024-01-26
Date of issue:2024-04-19
Advisor:Prof. Dr. Michaela Dippold
Referee:Prof. Dr. Michaela Dippold
Referee:Prof. Dr. Stefan Scholten
Referee:Prof. Dr. Sandra Spielvogel
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Abstract
English
Soil functions are key in all ecosystems globally. Specifically soils play a pivotal role in ecosystem biogeochemical cycles, sequestering but also mineralizing carbon (C), and in this context releasing nutrients from the soil organic matter (SOM). This thesis aims to improve our understanding of key soil microbial functions involved in C and nutrient cycling, starting with a case study from a grassland ecosystem where degradation induces massive C losses and by that shifts in C and nutrient stoichiometry, which induce cascading effects of the interaction of microbes and plants with C and nutrient cycling. This relation of key microbial functions to element stoichiometry is then further deepened in a laboratory study to generate a more holistic understanding on the control of the microbial metabolism by C and nutrient availability. About 2.5 % of global soil organic carbon (SOC) are stored in Tibetan Plateau’s Kobresia pygmaea grasslands. Topsoil degradation due to climate change and overgrazing with life stock led to a substantial loss in SOC and nitrogen (N), both vital elements for the flora and fauna. The erosion and depletion of C sources due to decreased C input and increased C mineralization forced a shift of the taxonomic composition of the microbial community and in consequence the enzymatic activities expressed. This led to a change in microbial functions from the utilization of hydrolytic enzymes towards enzymes capable of oxidizing the remaining recalcitrant SOM, being the initial step of the second phase of degradation of the pasture’s root mats and thereby removing Kobresia’s ability to further grow on the degraded sites. As the key switch towards oxidizing enzymes is controlled by microbial C and nutrient deficiency, further laboratory experiments were needed to elucidate the response of the microbial metabolism on such stoichiometric shifts. Especially deepened insights into the base C metabolism help to understand changes in soil microorganisms’ growth modes, the key feature of predicting their C and nutrient demand: (1) Under which conditions is the available soil C sufficient to invest into the formation of new biomass? (2) How does a natural soil microbial community react when C sources are scare or plentiful? (3) Under which conditions do they “merely” invest into the formation of storage compounds and not into growth? (4) How does nutrient availability in this context interact with C availability? To answer these research questions, it is necessary to also understand the regulatory processes from the transcription of base C metabolism genes towards the translation to actual proteins and to trace the C fluxes of the soil microbial metabolism. The samples from the field experiment in Tibet were analyzed using terminal restriction fragment length polymorphism (t-RFLP) and Illumina MiSeq sequencing to elucidate the microbial community structure. In addition, the enzyme activity of SOM degrading enzymes and N cycling related exoenzymes were measured. The research question regarding regulatory processes in the C metabolism would have been impossible to be answered in a field experiment, therefore the key questions were targeted by a laboratory experiment with natural agricultural soil. Under controlled laboratory conditions it was possible to manipulate the glucose amounts by adding low and high concentrations of glucose and N and phosphate (P) as nutrients. Over the course of the experiment of 96 h, microcosms were harvested after 24 and 96 hours for the measurements of phospho- (PLFA) and neutral lipid fatty acids (NLFA), polyhydroxybutyrate (PHB), microbial biomass carbon (MBC), the metatranscriptome and metaproteome. For 13C flux modelling, the glucose treatment solutions were also treated with uniformly and position-specifically labelled glucose and 13CO2 respiration was measured in time intervals. The degradation of the Kobresia pastures and the loss of the topsoil layer led to a shift in the microbial community structure especially in their mycorrhizal partners. As Kobresia pygmeae was first associated with arbuscular mycorrhizal fungi, the increasing severity of the degradation then led to a shift towards an association of Kobresia with ectomycorrhizal fungi. Furthermore, the bacterial community shifted towards species capable of degrading complex SOM and nitrifying bacteria further depleting the nutrient stocks on the plateau. The underlying regulatory change in microbial C metabolism of this shift were revealed by the laboratory experiment with reduced C availability in the agricultural soil: Metabolic flux modelling suggests that C fluxes were primarily directed into the catabolic glycolysis rather than the anabolic pentose phosphate pathway (PPP) in low glucose concentrations. After glycolysis, the C fluxes from acetyl-CoA usually enter the strongly catabolic tricarboxylic acid cycle (TCA), where the most energy can be generated. However, flux modelling also detected that from acetyl-CoA the C fluxes were directed towards storage compound formation in low glucose conditions. This hints towards a reserve storage strategy by the microbial community, which was supported by triacylglycerols (TAG) storage compound extraction. In contrast, the direct measurement of the storage compound metabolite PHB revealed a strong contribution to the formation of biomass under glucose excess conditions, a surplus storage strategy. So that both storage compounds fulfill different roles in microbial communities’ growth modes. The strong negative relationship between the TCA cycle and storage pathways in the metatranscriptome and metaproteome also underlined the importance of storage compound formation, which is a C resource of intermediate microbial availability, not contributing to the long-term SOC storage of an ecosystem. In conclusion, environmental changes such as climate change or anthropogenic induced degradation often exert unfavorable consequences on the soil microbial communities by shifting their structural composition and their functioning with implication on various scales. C availability and contents play a major role rather than other nutrients in the regulatory processes on the post-genomic levels, as C is the most restrictive growth factor in soils for bacteria and fungi. The formation of storage compounds seems to play a much bigger role in soil C cycling than anticipated before. Well-established molecular biology methods on the genome level can give a broad overview of the microbial community structure and – by some extend – of their potential function, but only the combination of metatranscriptomics, metaproteomics and meta-fluxomics in multi-meta-omics approaches will help broaden the insight into the microbial metabolism and deepen the understanding of microbial responses on environmental changes. However, transcription, translation, and the direction of C fluxes towards energy production, replicative growth or storage compound biosynthesis are not always coherent between the post- genomic levels. Therefore, the interpretation of regulatory processes remains a complex and difficult endeavor in natural microbial environments but harbor the possibility of a more profound understanding of the microbial C metabolism if further deepened in comparative, time-resolved approaches. By that, microbial feedbacks within degradation cascades can be revealed and potential mitigation strategies elaborated.
Keywords: Tibet; C Metabolism; Metatranscriptomics; Metaproteomics; Carbon flux modelling; Storage compounds; PHB; TAG; Glycolysis; Pentose phosphate pathway; TCA; Microbial community; Enzyme activity; SOC quality; Degradation; Pasture; Grassland; Erosion; Carbon cycle; Element cycles; Microbial ecology; Stable isotope analysis