Open microfluidics: droplet microarrays as next generation multiwell plates for high throughput screening.

Multiwell plates are prominent in the biological and chemical sciences; however, they face limitations in terms of throughput and deployment in emerging bioengineering fields. Droplet microarrays, as an open microfluidic technology, organise tiny droplets typically in the order of thousands, on an accessible plate. In this perspective, we summarise current approaches for generating droplets, fluid handling on them, and analysis within droplet microarrays. By enabling unique plate engineering opportunities, demonstrating the necessary experimental procedures required for manipulating and interacting with biological cells, and integrating with label-free analytical techniques, droplet microarrays can be deployed across a more extensive experimental domain than what is currently covered by multiwell plates. Droplet microarrays thus offer a solution to the bottlenecks associated with multiwell plates, particularly in the areas of biological cultivation and high-throughput compound screening.

Marine Fungi Select and Transport Aerobic and Anaerobic Bacterial Populations from Polycyclic Aromatic Hydrocarbon-Contaminated Sediments.

The organization of microbial communities in marine sediment relies on complex biotic and abiotic interactions. Among them, the interaction between fungi and bacteria plays a crucial role building specific microbial assemblages, resulting in metabolic networks adapted to environmental conditions. The fungal-bacterial interaction (FBI) includes bacterial translocation via fungal mycelia, allowing bacterial dispersion, and ecological niche colonization. In order to demonstrate that the translocation of bacteria through fungal mycelia involves bacterial selection, the mycelia of two fungi isolated from marine coastal sediment, Alternaria destruens F10.81 and Fusarium pseudonygamai F5.76, showing different strategies for uptake of polycyclic aromatic hydrocarbon (PAH), homogenous internalization and vacuole forming respectively, were used to translocate bacteria through hydrophobic hydrocarbon contaminated sediments. A. destruens F10.81 selected four specific bacteria, while bacterial selection by F. pseudonygamai F5.76 was not evident. Among the bacteria selected by A. destruens F10.81, Spirochaeta litoralis, known as strictly anaerobic bacterium, was identified, indicating that A. destruens F10.81 selects and transports both aerobic and anaerobic bacteria. Such a result is consistent with the observed formation of anoxic micro-niches in areas surrounding and affected by fungal hyphae. Our findings provide new insights on the selection and dispersion of bacterial communities by fungi, which are crucial for the organization of microbial communities and their functioning in coastal PAH-contaminated sediments. IMPORTANCE The study provides advances for understanding fungal-bacterial relationships, particularly on the selection and dispersion of bacterial communities by fungi, which are crucial for the organization of microbial communities and their functioning in coastal PAH-contaminated sediments. The transportation of bacteria via fungal hyphae (fungal highway) results in bacterial selection; in particular, fungal hyphae offer adequate conditions for the transport of both aerobic and anaerobic bacteria through hydrophobic patches for the colonization of novel niches.

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.

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.

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.

Enhanced biodegradation of PAHs in historically contaminated soil by M. gilvum inoculated biochar.

The inoculation of rice straw biochar with PAH-degrading Mycobacterium gilvum (1.27 × 1011 ± 1.24 × 1010 cell g-1), and the subsequent amendment of this composite material to PAHs contaminated (677 mg kg-1) coke plant soil, was conducted in order to investigate if would enhance PAHs biodegradation in soils. The microbe-biochar composite showed superior degradation capacity for phenanthrene, fluoranthene and pyrene. Phenanthrene loss in the microbe-biochar composite, free cell alone and biochar alone treatments was, respectively, 62.6 ± 3.2%, 47.3 ± 4.1% and non-significant (P > 0.05); whereas for fluoranthene loss it was 52.1 ± 2.3%; non-significant (P > 0.05) and non-significant (P > 0.05); and for pyrene loss it was 62.1 ± 0.9%; 19.7 ± 6.5% and 13.5 ± 2.8%. It was hypothesized that the improved remediation was underpinned by i) biochar enhanced mass transfer of PAHs from the soil to the carbonaceous biochar "sink", and ii) the subsequent degradation of the PAHs by the immobilized M. gilvum. To test this mechanism, a surfactant (Brij 30; 20 mg g-1 soil), was added to impede PAHs mass transfer to biochar and sorption. The surfactant increased solution phase PAH concentrations and significantly (P < 0.05) reduced PAH degradation in the biochar immobilized M. gilvum treatments; indicating the enhanced degradation occurred between the immobilized M. gilvum and biochar sorbed PAHs.