Impact of Fungal Hyphae on Growth and Dispersal of Obligate Anaerobic Bacteria in Aerated Habitats.

Anoxic microsites arising in fungal biofilms may foster the presence of obligate anaerobes. Here, we analyzed whether and to which degree hyphae of Coprinopsis cinerea thriving in oxic habitats enable the germination, growth, and dispersal of the obligate anaerobic soil bacterium Clostridium acetobutylicum. Time-resolved optical oxygen mapping, microscopy, and metabolite analysis revealed the formation and persistence of anoxic circum hyphal niches, allowing for spore germination, growth, and fermentative activity of the obligate anaerobe in an otherwise inhabitable environment. Hypoxic liquid films containing 80% ± 10% of atmospheric oxygen saturation around single air-exposed hyphae thereby allowed for efficient clostridial dispersal amid spatially separated (>0.5 cm) anoxic sites. Hyphae hence may serve as good networks for the activity and spatial organization of obligate anaerobic bacteria in oxygenated heterogeneous environments such as soil. IMPORTANCE Although a few studies have reported on the presence of anoxic microniches in fungal biofilms, knowledge of the effects of fungal oxygen consumption on bacterial-fungal interactions is limited. Here, we demonstrate the existence and persistence of oxygen-free zones in air-exposed mycelia enabling spore germination, growth, fermentative activity, and dispersal of the obligate anaerobe. Our study points out a previously overlooked role of aerobic fungi in creating and bridging anoxic microniches in ambient oxic habitats. Air-exposed hyphae hence may act as a scaffold for activity and dispersal of strictly anaerobic microbes. Given the short-term tolerance of strict anaerobes to oxygen and reduced oxygen content in the mycosphere, hyphae can promote spatial organization of both obligate anaerobic and aerobic bacteria. Such finding may be important for a better understanding of previously observed co-occurrences of aerobes and anaerobes in well-aerated habitats such as upland soils.

pH Distribution along Growing Fungal Hyphae at Microscale.

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing hyphae of the basidiomycete Coprinopsis cinerea. Time-lapse microscopy analysis of ratiometric fluorescence signals of >2400 individual bioreporters revealed an overall pH drop from 6.3 ± 0.4 (n = 2441) to 5.0 ± 0.3 (n = 2497) within 7 h after pH bioreporter loading to hyphal surfaces. The pH along hyphal surfaces varied significantly (p < 0.05), with pH at hyphal tips being on average ~0.8 pH units lower than at more mature hyphal parts near the entrance of the microfluidic observation chamber. Our data represent the first dynamic in vitro analysis of surface pH along growing hyphae at the micrometre scale. Such knowledge may improve our understanding of spatial, pH-dependent hyphal processes, such as the degradation of organic matter or mineral weathering.

Illuminate the hidden: in vivo mapping of microscale pH in the mycosphere using a novel whole-cell biosensor.

The pH of an environment is both a driver and the result of diversity and functioning of microbial habitats such as the area affected by fungal hyphae (mycosphere). Here we used a novel pH-sensitive bioreporter, Synechocystis sp. PCC6803_peripHlu, and ratiometric fluorescence microscopy, to spatially and temporally resolve the mycosphere pH at the micrometre scale. Hyphae of the basidiomycete Coprionopsis cinerea were allowed to overgrow immobilised and homogeneously embedded pH bioreporters in an agarose microcosm. Signals of >700 individual cells in an area of 0.4 × 0.8 mm were observed over time and used to create highly resolved (3 × 3 µm) pH maps using geostatistical approaches. C. cinerea changed the pH of the agarose from 6.9 to ca. 5.0 after 48 h with hyphal tips modifying pH in their vicinity up to 1.8 mm. pH mapping revealed distinct microscale spatial variability and temporally stable gradients between pH 4.4 and 5.8 over distances of ≈20 µm. This is the first in vivo mapping of a mycosphere pH landscape at the microscale. It underpins the previously hypothesised establishment of pH gradients serving to create spatially distinct mycosphere reaction zones.