Emiliania huxleyi, a calciferous microalga of global importance, is one of the major calcite producers in the world’s oceans. It forms calcium carbonate discs that are barely discernible under a light microscope. This biogenic calcite differs from fossil calcite primarily by its filigree three-dimensional structure. Ioanna Hariskos and other scientists from the Karlsruhe Institute of Technology (KIT) produce highly complex biogenic calcite particles and study their biomineralisation. Due to their unique physico-chemical properties, the small calcium carbonate discs called coccoliths are of major importance for industrial applications. In a cooperative project entitled “Cell-free biomineralisation with calcium carbonate being used as an example: a way towards in-vitro synthesis of highly structured composite materials”, the researchers are investigating how the process of biogenic calcification can be transferred to cell-free systems.
Algae produce many substances, including dyes, unsaturated fatty acids, polysaccharides and antioxidants that are a viable source of materials for the pharmaceutical and food industries. However, algae are still barely used as natural raw material sources. Emiliania huxleyi is a single-celled red microalga found in nearly all oceans, drifting in the upper, sunlit layers. Emiliania huxleyi is just one of 5,000 or so different species of phytoplankton, but it accounts for up to 80% of all photosynthetic plankton. It is one of the most interesting organisms in our oceans due to its ability to bind considerable amounts of carbon dioxide by producing calcite discs through photosynthesis*. It also makes a considerable contribution to our ecosystem and is an extremely interesting research object for (marine) scientists. These calcite-forming algae (calcite: crystalline form of lime) also created the beautiful white cliffs of Dover (GB) and the island of Rügen (D) which are largely made up of coccoliths.
*This process, which is also referred to as organic carbon pump, leads to a reduction in CO2 concentration in the upper layers of the ocean, resulting in the sequestration of CO2 from the atmosphere (ed. note: according to information provided by the Alfred Wegener Institute).
Ioanna Hariskos, PhD student at the KIT Institute of Process Engineering in Life Sciences, Bioprocess Engineering Division, studies Emiliania huxleyi microalgae from a somewhat different perspective. She is more interested in the application potential of the white calcite structures than their aesthetic side: "The biogenic calcite particles produced by the algae have a complicated three-dimensional structure that only nature can produce. The particles cannot be obtained by grinding limestone or by the precipitation of lime milk. This is why algal calcite particles have extraordinary potential for innovative industrial applications," says the biotechnologist, who did part of her postgraduate training at the University of Uppsala.
On the surface of each of these single-celled organisms are 15 to 120 calcite discs known as coccoliths, with highly filigree structures. The coccoliths are around 3 µm in size and resemble flat, fine sink strainers connected by a central tunnel.
The individual crystals interlock, forming compact, robust structures. The coccoliths are the result of controlled crystal growth in the centre of the alga. This biomineralisation is a highly organised process. "In contrast to fossil calcite, the highly complex calcite discs have extraordinary three-dimensional shapes and specific chemical and physical properties," says the scientist.
Due to its diverse properties, fossil calcite has always been used in a broad range of industries. "In the search for innovative products, we are looking for new, previously unknown properties. As the complicated irregular structures of coccoliths can only produced by nature, we came up with the idea of using microalgae to produce these calcite particles," says Ioanna Hariskos highlighting the objective of the cooperative project "ZeBiCa² - cell-free biomineralisation with calcium carbonate being used as an example". The KIT project is funded by the German Ministry of Education and Research (BMBF).
A suitable cultivation method that generates large quantities of coccoliths is the prerequisite for the innovative characterisation of the calcite particles for industrial application and cell-free synthesis. "To start with, we had to find out what the cells needed in order to be able to form biominerals." As part of the BMBF-funded project, Hariskos developed a cultivation method that led to high coccolith yields.
Algae strains can differ considerably in terms of their morphological structure. The researchers therefore selected a strain with a high degree of calcification. The individual crystals of the chosen strain are so close together that no gaps can be discerned. Instead, the coccoliths of this particular strain form a closed shell around the cell. "Developing a cultivation method that enabled us to grow large cell numbers under artificial conditions was a huge challenge," says the scientist who did a master's degree in biotechnology with a special focus on process engineering at RWTH Aachen University.
"Growing cells at a higher density than in their natural environment, i.e. seawater, means that the algal cells can influence each other; the higher than normal coccolith density can also have a disturbing effect." The researchers had to develop optimal process conditions for producing high yields whilst also ensuring that they would not damage the crystals. Hariskos and her team therefore used modified artificial seawater as a substrate. The algae also require light for photosynthesis. "We are using a special LED technology that was developed at the KIT and which has been used around the world for quite some time. This LED enclosure produces less heat than halogen or other light sources," says Hariskos highlighting the advantages of the light-emitting diodes developed at the KIT.
In the bioreactor, the algae performing photosynthesis can thus be exposed to light for 24 hours a day and the light intensity adapted to the respective culture density. The researchers optimised the cultivation process by varying different process parameters in different bioreactors. Parameters such as cell density, total alkalinity and the concentration of dissolved inorganic carbon and salts were analysed daily.
"In nature, from 1,000 to a maximum of 100,000 cells of the small calcite former are found in 1 ml seawater, whereas we succeeded in cultivating up to 100 million cells per millilitre, which is a sensationally high yield," Hariskos says. The successful development of a valid production process in a 2l bioreactor with sophisticated measurement and control systems and under defined conditions represents the milestone of the last two years.
"We achieved cell densities that were 10,000 to 100,000 times higher than ever before, leading to coccolith yields in the g/l scale." This is a remarkable achievement, considering that our industrial partners that use coccoliths as colouring agents need huge quantities of the material. In order to be able to characterise in greater detail these largely unexplored particles that have huge innovative potential, as well as enable large-scale production, Hariskos plans to increase the process scale and integrate an appropriate separation process.
This would then allow the researchers to characterise the calcite discs in greater detail using scanning electron microscopy, white balance values, specific surface area values, pore size distribution values and chemical analyses. Application of the coccoliths in the colouring and surface-treatment industry depends upon knowing parameters such as particle size, specific surface and white balance values. Hariskos was able to produce white coccolith powder with a slight yellow tinge. The potential use of the coccoliths for application in paints has yet to be tested. Due to their defined size and the extraordinary filigree structure, coccoliths have the potential to be used for modifying colour and glazing properties.
Another area of interest for industrial application is the use of calcite nanoparticles for producing abrasive or other special papers. "The chemical composition of coccoliths can potentially be influenced, and used to specifically adjust the resulting mechanical properties such as abrasiveness and scrub resistance, according to the application they will be used for," says Horiska referring to the innovative potential of coccoliths. "The calcite particles also have the extraordinary ability to split light into two beams. The birefringence of calcite could therefore potentially be applied in optics. However, this requires the particles to be extraordinarily pure. As they contain biological, organic material, further scientific work is needed to explore whether and how the required purity can be achieved. "The biogenic particles have an as yet unexploited potential, including for medical applications," says the scientist.
By producing a large quantity of biogenic calcite particles, Horiska has paved the way for future upscaling and characterisation in cooperation with industrial partners. If the researchers manage to acquire funds for a follow-up project, Hariskos and her team will be able to turn these promising scientific plans into reality.