In the tiny, beaded chain of the cyanobacterium Anabaena sp. ATCC 33047, two different cells, the photosynthetic factory worker and the nitrogen-fixing specialist, play distinct and powerful roles in creating solid minerals. 

A team led by Renewable And Sustainable Energy Institute (RASEI) Fellow Jeff Cameron and Nature, Environment, Science & Technology (NEST) Studio co-founder Erin Espelie, used advanced high-resolution microscopy to capture the key moments; the factory worker leaks materials when stressed, and the specialist accelerates crystal growth through contact, proving that single-cell behaviors are a vital trigger for biomineralization. Understanding the cellular processes could inform large-scale applications, from oceanic buffering and soil improvement to mineral formation, and living building materials that sequester carbon.

A central enabling technology to lower pollution and reduce carbon emissions is developing clever ways to capture, and handle carbon dioxide. One avenue of investigation is to use processes already developed by Nature. There is significant research focused on using one of the Earth’s oldest and powerful processes: Microbiologically Induced Calcium Carbonate Precipitation, or MICP for short. Bacteria and algae through their normal life functions naturally create rock, specifically calcium carbonate, the main component of limestone. This process is a critical process in oceanic buffering and holds immense potential promise for green technologies. If we can understand, and harness this process, we could use such bacteria for a broad range of applications. We could create “living” cements for self-healing concrete, stabilize fragile soils, even enhance industrial carbon dioxide sequestration. However, to control this process we first need to understand the specific cellular blueprints that guide these microbial construction projects. Until now, those blueprints have been frustratingly fuzzy.

To better understand the puzzle of biomineralization the team explored the cellular structure of the cyanobacteria Anabaena sp. ATCC 33047 (hereafter Anabaena). Think of this organism as a tiny “Filament Factory”, one that grows as a string of cells, essentially a beaded green chain (they show up as red in the images because of the microscopy technique), where labor is divided in specific jobs. The links in the chain are not identical, it contains two specialized cell types that perform distinct, but equally important tasks.

First, let’s consider the Vegetative Cells, which are like tireless “Photosynthetic Factory Workers”. These are the green, abundant cells with the primary job of harvesting solar energy to convert carbon dioxide into sugars (Photosynthesis). This process has long been proposed as the main cause for triggering rock formation through MICP, as it raises the local pH, making the environment more alkaline, which encourages calcium carbonate to precipitate.

The other kind of cells, which can be found scattered along the filament, are called Heterocysts. These are like “Nitrogen-Fixing Specialists”. These cells are slightly larger, more solidly built, and specialize in converting atmospheric nitrogen gas into a usable form for the entire filament. This requires an extremely lo-oxygen environment, distinguishing the heterocysts and giving them a significant influence over the cells surrounding chemical environment.

To understand the process in a stepwise fashion the team were able to treat the bacterial system with a specific nutrient cocktail that essentially “turned off” the generalized photosynthesis-driven precipitation and instead focus solely on the effects of these two specialized cells. By developing approaches to shutdown specific parts of the process the team could use advanced microscopy techniques to better pin-point the single-cell behaviors responsible for triggering the formation and growth of microscopic rock.

Unlocking this level of detail in the cellular workings of a cyanobacteria requires specialized tools. The researchers used a suites of powerful high-resolution techniques to interrogate the bacteria, including Quantitative Fluorescence Microscopy and Raman Microscopy, that enabled them to watch the action unfold. The ability to directly observe the single-cell processes was critical to determining how the “Filament Factory” uses two distinct mechanisms for biomineralization.

The first observation centers around the Vegetative Cells, or the “Photosynthetic Factory Workers”. While the cells are usually busy using solar energy to capture carbon dioxide the high-resolution microscopy captured what happens when these cells are under mechanical stress, such as when they are bent by other cells, or squashed against an existing mineral structure. The team were able to watch in real-time as this physical pressure caused the cells membrane to rupture. This breach of the membrane releases, or leaks, a key chemical, the sequestered inorganic carbon (bicarbonate) that the cell was holding inside. This rapid, localized surge of carbon creates excellent conditions for the formation of a new crystal at the leakage site. This reframes the start of the process. It is not just a passive gradual change in the environment that causes crystal growth, instead it can be caused by an active, stress-induced cell failure that is a trigger for calcite crystal nucleation.

The second observation concerns the actions of the Heterocyst Cells, or the “Nitrogen-Fixing Specialists”. Using the powerful techniques that enabled the researchers to peer into the inner workings of the cells the team were able to confirm that when a heterocyst cell came into direct contact with an existing calcite crystal “seed”, the crystal experienced rapid and dramatic growth. Crucially, this accelerated growth did not happen when a vegetative cell touched the crystal.

The team proposes that this dramatic crystal growth is connected to the function of Heterocyst Cell. Nitrogen fixation is a chemical transformation that consumes protons (H⁺). By pulling these protons out of the surrounding water, the heterocyst locally, and rapidly, increases the pH (alkalinity) of the microenvironment, which is amplified at the point of contact. This sudden shift in pH provides ideal conditions to effectively “glue” dissolved ions onto the existing crystal, resulting in rapid growth.

These findings describe how these two specialized cells have complementary roles. One is the nucleation trigger when stressed, and the other is the growth accelerator when in contact.

This detailed observation and analysis of the processes happening at the single-cell level shifts our understanding around the processes involved in biomineralization. Instead of thinking of microbial rock formation as a slow and uniform chemical reaction driven by large-scale phenomena like photosynthesis, this work illustrates mechanisms that are controlled and function-specific processes that are dictated by the precise cellular roles and localized behavior of individual cells.

The understanding building from these findings has the potential to inform a wide-range of applications. By isolating the “stress leak” trigger in vegetative cells and the growth accelerator from the heterocysts, researchers could design systems that intentionally apply mechanical stress, triggering crystal formation and accelerating the growth of carbon dioxide sequestering materials. This could have application in oceanic buffering and technologies for bio-concrete and soil rectification.

The development and application of advanced microscopic techniques has provided the bio-engineering world a new set of variable that they can use in bacterial engineering. By moving from a vague knowledge of “microbes make rock”, to a precise understanding of how the “Filament Factory” uses specialized cells to build, and grow, calcite crystals, the field is a step closer to harnessing this powerful natural approach for using carbon dioxide in a cleaner, more efficient way.