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Looking Inside a Biofilm: Microprofiling with Miniaturized Sensors

Biofilms and microbial granules are highly structured microbial communities in which microorganisms experience very different chemical conditions over distances of only a few hundred micrometres. Oxygen, pH and electrical potential can change dramatically from the outer surface toward the interior of a granule, reflecting the combined activity of different microbial populations and their metabolic processes.

To investigate these microscale gradients, our project uses microprofiling with miniaturized sensors. This approach allows us to measure chemical and electrochemical conditions directly inside microbial granules, providing a detailed view of the environments in which microorganisms live and interact.

Measuring microscale gradients

The principle of microprofiling is relatively simple: a very small sensor is progressively moved through a microbial granule while measurements are continuously recorded. Because the sensor tip is only a few micrometres in size, it can resolve changes in environmental conditions at a spatial scale that would not be possible using conventional electrodes.

In our experiments, we measured depth profiles of three key parameters:

• Electric potential (EP), which provides information about the electrochemical environment within the granule.

• pH, an important indicator of microbial activity and chemical transformations.

• Dissolved oxygen (O₂), which reveals how oxygen is transported into and consumed within the microbial community.

Together, these measurements provide a multidimensional picture of the conditions experienced by microorganisms at different depths.

Controlling oxygen conditions

Oxygen availability is particularly important when studying microbial granules because microorganisms consume oxygen as it diffuses from the surrounding water toward the interior. To investigate these gradients under controlled conditions, the oxygen concentration of the bulk water was adjusted by flushing the aquarium with a controlled gas mixture of N₂ and CO₂. Gas flow was regulated using a gas mixer, with a total gas flow of approximately 315 mL min⁻¹. The oxygen concentration in the surrounding water was continuously monitored using a fiber-optic oxygen sensor connected to an O₂-reader .

This control of the bulk oxygen concentration is essential because it provides a defined starting condition from which oxygen penetration and consumption within the granule can be investigated.

Moving the sensor through the granule

The key component of the methodology is the miniaturized sensor itself. The microsensor is positioned above the granule and carefully advanced through its depth, allowing the parameter of interest to be measured at successive positions.

A stereomicroscope was used throughout this procedure to visualize the granule and precisely identify the point at which the microsensor tip entered the microbial structure. This visual control is particularly important because the sensor must enter the granule at a known location while avoiding damage to the structure as much as possible.

As the sensor moves from the surrounding water toward the interior, the recorded signal generates a depth microprofile. The resulting profile can reveal, for example, where oxygen begins to decline, where pH changes most strongly, or how the electric potential varies between the outer and inner regions of the granule.

Before measurements, the microsensors were calibrated using established procedures. Vertical sensor movement was precisely controlled, and measurements were acquired continuously during the profiling process.

From profiles to microbial activity

The resulting microprofiles are more than measurements of concentration or potential, they provide insight into the spatial organization and activity of the microbial community.

For example, an oxygen profile can indicate the depth to which oxygen penetrates before being depleted by microbial respiration. A corresponding pH profile may reveal regions where microbial metabolism produces or consumes protons. Electric-potential measurements provide complementary information about changes in the local electrochemical environment.

By comparing these profiles under different experimental conditions, we can investigate how the microbial community responds to changes in its environment and how different processes are spatially organized within the granule.

Why microprofiling matters

Conventional measurements typically describe the average conditions in the surrounding water. Microprofiling takes us one step further by showing what happens inside the microbial community itself.

This distinction is crucial for biofilms and microbial granules. Two samples may have identical bulk-water conditions while containing very different microenvironments internally. A steep oxygen gradient, for instance, can create an oxygen-rich outer layer alongside oxygen-depleted regions deeper inside the granule. Such microscale heterogeneity can allow different microbial metabolisms to occur only micrometres apart.

Miniaturized sensors therefore act as a window into the hidden chemical architecture of microbial communities. By combining measurements of electric potential, pH and oxygen, our methodology helps reveal how physical and chemical gradients shape microbial activity and interactions within biofilms and granules.

In this project, these high-resolution measurements contribute to a better understanding of the mechanisms governing microbial communities and their performance in complex environments such as wastewater treatment systems.