Quantification of growth and nutrient consumption of bacterial and fungal cultures in microfluidic microhabitat models.

Understanding microbes in nature requires consideration of their microenvironment. Here, we present a protocol for quantifying biomass and nutrient degradation of bacterial and fungal cultures (Pseudomonas putida and Coprinopsis cinerea, respectively) in microfluidics. We describe steps for mask design and fabrication, master printing, polydimethylsiloxane chip fabrication, and chip inoculation and imaging using fluorescence microscopy. We include procedures for image analysis, plotting, and statistics. For complete details on the use and execution of this protocol, please refer to Arellano-Caicedo et al. (2023).1.

Habitat complexity affects microbial growth in fractal maze.

The great variety of earth's microorganisms and their functions are attributed to the heterogeneity of their habitats, but our understanding of the impact of this heterogeneity on microbes is limited at the microscale. In this study, we tested how a gradient of spatial habitat complexity in the form of fractal mazes influenced the growth, substrate degradation, and interactions of the bacterial strain Pseudomonas putida and the fungal strain Coprinopsis cinerea. These strains responded in opposite ways: complex habitats strongly reduced fungal growth but, in contrast, increased the abundance of bacteria. Fungal hyphae did not reach far into the mazes and forced bacteria to grow in deeper regions. Bacterial substrate degradation strongly increased with habitat complexity, even more than bacterial biomass, up to an optimal depth, while the most remote parts of the mazes showed both decreased biomass and substrate degradation. These results suggest an increase in enzymatic activity in confined spaces, where areas may experience enhanced microbial activity and resource use efficiency. Very remote spaces showing a slower turnover of substrates illustrate a mechanism which may contribute to the long-term storage of organic matter in soils. We demonstrate here that the sole effect of spatial microstructures affects microbial growth and substrate degradation, leading to differences in local microscale spatial availability. These differences might add up to considerable changes in nutrient cycling at the macroscale, such as contributing to soil organic carbon storage.

Habitat geometry in artificial microstructure affects bacterial and fungal growth, interactions, and substrate degradation.

Microhabitat conditions determine the magnitude and speed of microbial processes but have been challenging to investigate. In this study we used microfluidic devices to determine the effect of the spatial distortion of a pore space on fungal and bacterial growth, interactions, and substrate degradation. The devices contained channels differing in bending angles and order. Sharper angles reduced fungal and bacterial biomass, especially when angles were repeated in the same direction. Substrate degradation was only decreased by sharper angles when fungi and bacteria were grown together. Investigation at the cellular scale suggests that this was caused by fungal habitat modification, since hyphae branched in sharp and repeated turns, blocking the dispersal of bacteria and the substrate. Our results demonstrate how the geometry of microstructures can influence microbial activity. This can be transferable to soil pore spaces, where spatial occlusion and microbial feedback on microstructures is thought to explain organic matter stabilization.

Microfluidic chips provide visual access to in situ soil ecology.

Microbes govern most soil functions, but investigation of these processes at the scale of their cells has been difficult to accomplish. Here we incubate microfabricated, transparent 'soil chips' with soil, or bury them directly in the field. Both soil microbes and minerals enter the chips, which enables us to investigate diverse community interdependences, such as inter-kingdom and food-web interactions, and feedbacks between microbes and the pore space microstructures. The presence of hyphae ('fungal highways') strongly and frequently increases the dispersal range and abundance of water-dwelling organisms such as bacteria and protists across air pockets. Physical forces such as water movements, but also organisms and especially fungi form new microhabitats by altering the pore space architecture and distribution of soil minerals in the chip. We show that soil chips hold a large potential for studying in-situ microbial interactions and soil functions, and to interconnect field microbial ecology with laboratory experiments.