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NanoSIMS Enabled Approach to Understand Bacterial and Fungal Cellulose Degraders in Soils

Final Report Summary - SAE_SNSP_UVA (NanoSIMS Enabled Approach to Understand Bacterial and Fungal Cellulose Degraders in Soils)

Please refer to the attachment.
Terrestrial ecosystems encompass a large area on Earth, yet our understanding of the microbial diversity, activity and factors governing these microbial activities mere centimeters beneath our feet is rudimentary at best. One such area that urgently demands a better understanding of the contributors and mechanisms is the terrestrial carbon cycle. Mineral soils contain the largest pool of carbon on Earth; cellulose is one of the major constituents of this carbon pool since it is a key component of plant structural carbon. Members of the Bacteria and Fungi are essential for degrading cellulose in several ecosystems and thus are essential for cycling carbon. The deconstruction of cellulose is accomplished through the synergistic interaction of a suite of enzymes - cellulases (endo-β-1,4-glucanases) - converting the long polysaccharide cellulose to the monomer glucose. The majority of our knowledge on cellulose degradation has been restricted to organisms that we can grow in the laboratory along with subsequent analyses with standard activity assays. The reliance on these growth-based methods to understand soil function, such as cellulose degradation, can be misleading since only 0.1 to 1% of the total microbial community has been or can be cultivated. Thus, it is plausible to assume that we are missing a great deal of untapped cellulose degrading diversity through the historical reliance on cultivation methods. More recent molecular-based studies identified active cellulolytic Bacteria and Fungi across different soils. However these studies (1) did not delaminate the specific bacterial and fungal contributions to cellulose degradation, (2) did not identify drivers of cellulose degradation, and (3) did not reveal systematic differences in the time course of cellulose degradation among these active groups.
Dr. Eichorst has advanced our understanding of cellulose degradation and the factors that govern it during her Marie Curie International Incoming Fellowship. In addition, she developed and enhanced single-cell methods with which one can answer fundamental questions in terrestrial ecosystems. The scientific outcomes of this fellowship include: (1) describing temporal patterns across members of the Bacteria and Fungi to cellulose degradation in an Austrian beech forest soil; (2) identification of carbon substrate and nitrogen availability on the cellulose degradation process; and (3) development and optimization of single-cell techniques for soil investigations.
Recently methods were developed that allow the investigations of uncultured single microbial cells, such as single-cell genome sequencing to understand their genomic potential and high-resolution secondary ion mass spectrometry (NanoSIMS) or Raman microspectroscopy to understand their associated activity. To infer the single-cell activity, the latter two methods are often applied in combination with stable isotope tracer incubations. The application of these techniques proves to be particularly challenging in terrestrial environments due to the large background of organic and inorganic soil particles. Dr. Eichorst enhanced cell removal treatments in conjunction with density gradient to generate a cell fraction with reduced soil particle load and particles of small enough size to allow single-cell analysis by NanoSIMS or Raman microspectroscopy. She documented changes in the community structure using next generation sequencing (SSU amplicon – 16S rRNA gene). As a proof-of-concept, she was able to successfully apply this cell extraction method to detect cellulose-degrading microorganisms from an Austrian beech forest soil by NanoSIMS. The measured 13C-enrichments ranged from ca. 20 to 60 atom %. Dr. Eichorst also tested the possibility to detect 13C-enriched cells by Raman microspectroscopy based on the described “red shift” of the phenylalanine peak upon 13C-isotope incorporation from 13C-cellulose in soil microcosm experiments.
Dr. Eichorst used a combined biogeochemical, molecular, and enzymatic approach to characterize cellulose-degrading guilds over time in an Austrian beech forest soil. Carbon is cycled by an array of microorganisms in soils. It is believed that the utilization of labile carbon, as well as complex carbon, encompasses a great deal of functional redundancy across members of the Bacteria and Fungi, thus allowing this process to occur under varying environmental conditions. Nitrogen is also regulator of complex carbon degradation, since the production of complex carbon degrading enzymes requires nitrogen. She hypothesized that by varying certain edaphic properties (such as carbon and nitrogen) that can limit cellulose degradation, she could uncover different cellulose-degrading guilds of bacteria and fungi and elucidate their ecological niches. The addition of N in inorganic or organic forms significantly increased cellulolytic activity as measured by total 13CO2 production and cellulase activity. The cellulose-responsive bacterial and fungal communities were assessed with 13C-phospholipid fatty acid analysis (PLFA). The proportion of bacterial and fungal PLFAs did not significantly change within a tested treatment over time, however there were significantly higher proportions of extracted 13C-PLFAs from the nitrogen addition treatments. The ratio of assignable bacterial and fungal markers were calculated to ascertain the prevalence of bacteria and fungi across the treatments. The addition of glucose caused a community shift towards bacteria; where as the inclusion of inorganic nitrogen increased the fungal proportion. Dr. Eichorst is currently identifying the active microbial cellulose-degrading community through stable isotope probing and will subsequently apply her established single-cell sample preparation pipeline to investigate the cellulose-degrading activity of identified community members in the single-cell level by FISH-NanoSIMS.