Redefining the Subsurface Biosphere: Characterization of Fungi Isolated From Energy-Limited Marine Deep Subsurface Sediment

Abstract

The characterization of metabolically active fungal isolates within the deep marine subsurface will alter current ecosystem models and living biomass estimates that are limited to bacterial and archaeal populations. Although marine fungi have been studied for over fifty years, a detailed description of fungal populations within the deep subsurface is lacking. Fungi possess metabolic pathways capable of utilizing previously considered non-bioavailable energy reserves. Therefore, metabolically active fungi would occupy a unique niche within subsurface ecosystems, with the potential to provide an organic carbon source for heterotrophic prokaryotic populations from the transformation of non-bioavailable energy into substrates, as well as from the fungal necromass itself. These organic carbon sources are not currently being considered in subsurface energy budgets. Sediments from South Pacific Gyre subsurface, one of the most energy-limited environments on Earth, were collected during the Integrated Ocean Drilling Program Expedition 329. Anoxic and oxic sediment slurry enrichments using fresh sediment were used to isolate multiple fungal strains in media types that varied in organic carbon substrates and concentration. Metabolically active and dormant fungal populations were also determined from nucleic acids extracted from in situ cryopreserved South Pacific Gyre sediments. For further characterization of physical growth parameters, two isolates were chosen based on their representation of the whole South Pacific Gyre fungal community. Results from this study show that fungi have adapted to be metabolically active and key community members in South Pacific Gyre sediments and potentially within global biogeochemical cycles.

Keywords: IODP (Integrated Ocean Drilling Program) Expedition 329; fungi; marine sediment; oligotrophic; subsurface.

Figure 1

Sampling locations of IODP Expedition 329. The large dots represent the locations from which the two main isolates characterized in this study were derived. Figure was made using GeoMapApp (Ryan et al., 2009).

Figure 2

Microscopic images showing morphological characteristics of isolate SPG-F1 from U1371E-14H2 (A–H) and isolate SPG-F15 from U1368D-2H1 (I–P) grown on either potato dextrose agar (A–D,I–L) or marine broth agar (E–H,M–P).

Figure 3

Phylogenetic tree of 18S rRNA gene sequences from clones and isolates from the South Pacific Gyre sediments.

Figure 4

Radial growth rates (in mm) of all isolates on marine broth agar (MBA) or potato dextrose agar (PDA) under oxic or anoxic conditions. (A) Aerobic growth with MBA. (B) Anaerobic growth with MBA. (C) Aerobic growth with PDA. (D) Aerobic growth with PDA. Error bars represent the standard error calculated for 12 biological replicates.

Figure 5

Effect of temperature on SPG-F1 and SPG-F15 growth, measured in mg over time. (A) 4°C growth. (B) 10°C growth. (C) 15°C growth. (D) 21°C growth. (E) 26°C growth. Only 4°C growth has a control for each isolate because the growth analysis was done at different times. Error bars represent standard error calculated for 3 biological replicates.

Figure 6

Effect of salinity on SPG-F1 and SPG-F15 growth, measured in mg over time. (A) 0% NaCl growth. (B) 1% NaCl growth. (C) 2% NaCl growth. (D) 6% NaCl growth. (E) 8% NaCl growth. Time point 0 for 6 and 8% NaCl were removed as they were outliers. Error bars represent standard error calculated for 3 biological replicates.

Figure 7

Effect of pH on SPG-F1 and SPG-F15 growth, measured in mg over time. (A) pH 3 growth. (B) pH 6 growth. (C) pH 8 growth. Error bars represent standard error calculated for 3 biological replicates.

Figure 8

Growth of SPG-F1 and SPG-F15 on isotopically labeled lignin under oxic (A) and anoxic (B) conditions. Shown are accumulations of lignin derived ¹³C, expressed as δ¹³C-CO₂ vs. the Vienna PDB standard, in the pool of inorganic carbon over time.