Evaluating the impact of redox potential on the growth capacity of anaerobic gut fungi

Abstract

Anaerobic gut fungi (AGF, Neocallimastigomycota) inhabit the alimentary tract of herbivores. Although strict anaerobes, studies have suggested their capacity to retain viability after various durations of air exposure. It is currently unclear whether AGF can actively grow, and not merely survive, in redox potentials (E_h) higher than those encountered in the herbivorous gut. We evaluated the growth of two AGF strains (Orpinomyces joyonii and Testudinimyces gracilis) at various E_h levels, achieved by manipulating the concentrations of reductant (cysteine hydrochloride) in culture media. Both strains exhibited robust and sustainable growth at negative E_h (-50 mV or below). However, growth in the absence of cysteine hydrochloride (E_h value around +50 mV) was possible only for O. joyonii and only for one subcultivation. The capacity to grow at +50 mV was further confirmed in four additional taxa (Pecoramyces ruminatium, Anaeromyces mucronatus, Aklioshbmyces papillarum, and Piromyces communis), while two (Aestipascuomyces dupliciliberans and Capellomyces foraminis) failed to grow under these conditions. Our results establish the ability of AGF to grow at redox potential values higher than those encountered in their natural habitats. Such capability could contribute to efficient AGF dispersal and horizontal transmission between hosts, and could have important implications for industrial applications of AGF.

Keywords: anaerobic fungi; anaerobiosis; redox potential.

Figure 1.

Growth cycle of AGF. The possible existence of yet-unidentified resting stage/aerotolerant structures (depicted by a “?”) has been proposed but not conclusively demonstrated yet.

Figure 2.

Growth of AGF strains Orpinomyces joyonii strain AB.3 (left column) and Testudinimyces gracilis strain T130A.3 (right column) in media with 416 (A), 208 (B), or 0 mg/l (C) CystHCl. The bars in each graph correspond to the average (from four replicates) headspace gas pressure (ΔPSI) (primary Y-axis) used here as a proxy for growth (gray error bars showing the standard deviation), and the thick line corresponds to the average (from four replicates) redox potential E_h (secondary Y-axis) measured at the conclusion of each subculture (black error bars showing the standard deviation). The dashed line shows the cutoff headspace gas pressure value (ΔPSI = 4) we used to consider the growth positive. The number of subcultures are shown on the X-axis. The interval between subcultures was 4 days for Orpinomyces joyonii strain AB.3 and 7 days for Testudinimyces gracilis strain T130A.3.

Figure 3.

Photograph documenting the ability of Orpinomyces joyonii strain AB.3 to grow in both fully reduced media with 416 mg/l HCl (left), as well as non-reduced media without CystHCl. The orange color is formed due to the blending of yellow color of rumen fluid, and red/oxidized color of as evident by the oxidized (orange, a mix of yellow from rumen fluid and red, the color of the oxidized from of resazurin).

Figure 4.

Growth of AGF strains Pecoramyces ruminatium strain orc.30, Piromyces communis strain AP1.1, Anaeromyces mucronatus strain can.4, Aklioshbmyces papillarum strain wts.1, Aestipascuomyces dupliciliberans strain SR1.3, and Capellomyces foraminis strain Cap.2A in media with 0 mg/l CystHCl. The bars in each graph correspond to the pressure (ΔPSI) (primary Y axis) used here as a proxy for growth, and the thick line corresponds to the redox potential E_h (secondary Y axis) measured at the conclusion of each subculture. The dashed line shows the cutoff gas pressure value (ΔPSI = 4) we used to consider the growth positive. The number of subcultures are shown on the X-axis. The interval between subcultures was 4 days.