59th Annual Report on Research 2014

            Necromass of Fusarium avenaceum, a common temperate soil saprotroph, was placed in stainless steel (100μm mesh) packets and incubated in laboratory macrocosms and in the field. The field site was the Dixon Prairie of the Chicago Botanic Garden. The contents were analyzed via FTIR, thermochemolysis GC-MS using tetramethyl ammonium hydroxide (TMAH), and elemental analysis-isotope ratio mass spectrometry. Fungal material was rapidly lost as indicated by bulk C mass (Fig. 1). Approximately 75-80% of the Fusarium OM has a turnover time of ~ 1 week whereas ~10% has a potential turnover time exceeding several months.

Fig. 1: The loss of bulk Fusarium OC as a function of time in soil degradation experiments (lab and field incubations). Values are normalized to the time 0 benchmark. Turnover times (τ) are noted for different parts of the degradation curves.

            The selective removal of reactive fractions over time transforms the bulk and molecular composition of the fungal materials. As an example during the first few days of incubation, the increase in concentration of glucosamine, the monomer building block of chitin and chitosan, reflects the selective removal of more reactive compounds (Fig. 2). Loss of the chitin/chitosan is apparent thereafter. The parallel behavior of the d¹⁵N tracks the sequential removal of reactive N-containing compounds and chitin/chitosan and suggests that the N-isotopic composition of the chitin/chitosan is distinctly different (¹⁵N-depleted) from other nitrogenous components.

Fig. 2: d¹⁵N and glucosamine concentrations (C/C₀) in Fusarium necromass as a function of time of degradation in both laboratory and field experiments (n=3). Note that the d¹⁵N scale is inverted to better illustrate the parallel behaviors.

FTIR analysis also reveals changes in organic composition over time. The reduction in the absorbance of a C-O stretch centered at 1073 cm^-1 is likely due to the loss of a dominant carbohydrate, such a β-glucan or chitin/chitosan. Additional C-O containing components as well as amide functionality persist to 50 days, though with changes in their relative abundance. Insofar as chitin appears to be effectively removed during degradation (Fig. 2), we conjecture that the residual material may be a cross-linked cell wall glycoprotein or its diagenetically altered product. Changes in the relative concentrations of methylated hexoses derived from TMAH-thermochemolysis also indicate preferential loss of some carbohydrates.

Long-chained n-fatty acids exhibit a range of reactivities despite their similar structures (Fig. 3). The order of reactivity is C20:1> C18:1>C16> >C18>C24. Notable quantities of the fatty acids persist to the end of the experiment. We hypothesize that the fatty acids reside in and/or are bound to biochemical matrices with varying turnover times.

Fig. 3: Fatty acid concentrations in degrading Fusarium as a function of time in laboratory degradation experiments.

Fig. 4: Fatty acid and sterol concentrations in degrading Fusarium as a function of time from the laboratory experiments.

Other lipids exhibit a more complex behavior (Fig. 4). The fungal biomarker ergosterol and an unidentified sterol decrease rapidly at first and then peak at days 8-12. The n-fatty acids C16:1 and C18:1 (a different isomer than that depicted in Fig. 4) reach maximum concentrations at days 4 and 8 respectively and then decrease. Fungi contain sterol-rich domains, such as chitisomes, that are perhaps concentrated in the early stages of degradation and then decomposed. Alternatively, colonization of the Fusarium OM via other soil fungi may be contributing to the lipids. Microscopic and SEM analyses of the residue do indicate hyphae and spores from other fungi however the bulk of the material appears to be the Fusarium residue.

The tentative conclusion reached from these data is that some lipid fractions may persistent long enough to contribute long-term sequestration, especially if cross-linking or other diagenetic reactions render them biologically unreactive. We are currently analyzing samples that used other fungal species and are yielding fundamentally the same results. The identity of the persistent macromolecular material that is serving as a host for the lipids is also being sought. The role of fungi in the sequestration of C in soils and sediments has barely been considered, thus this study should open new avenues of research in the field.

This project has allowed me to move into several new areas of research involving microbial cycling in soils, the contribution of fungi to the C-cycle and the use of biomarkers in organic geochemistry. This has led to proposal submissions to NSF and DOE. This was a new direction as well for the post-doctoral fellow supported by the project, Kathryn Schreiner, who plans to continue this work as an assistant professor at the University of Minnesota – Duluth. This project has also provided research opportunities for undergraduate students. Jessie Moravek is studying the potential role of chitin as a source of bound fatty acids. Aarohi Shah is studying lipid production by chitinolytic fungi. Both students have had to do extensive method development and have been remarkably successful. We have also hosted a REU student from the Chicago Botanic Garden (Ben Sedillo) this summer who began studies with additional fungal species. He was introduced to fungal biochemistry and FTIR analyses for the first time.