The impact of nutrients on microalgae growth and what this means for biological models

Depending on the strain, microalgae are rich in protein, carbohydrates, lipids and offer highly valuable components such as astaxanthin or β‐carotene. Moreover, microalgae take up CO2 and can grow solely on sunlight with high areal productivities. Currently, microalgae biomass is mainly produced for high‐value applications related to human consumption, including nutraceuticals and pharmaceuticals. However, microalgae also showed potential for other applications e.g. for bulk chemical production, wastewater treatment and aqua feed. High production costs of 5 to 50€ per kg make a wider application spectrum unfeasible, which is partly due to the significantly lower production rates on industrial scale compared to lab‐scale. To solve this problem a better understanding of interactions between the different growth input parameters is required.

Specifically, the carbon, nitrogen and phosphorous metabolisms play a key role in the growth of microalgae. Nitrogen is required to synthesize protein, RNA and DNA, while phosphorous is essential to RNA, DNA, ATP and phospholipid synthesis. Since protein structures are omnipresent in every aspect of the cell such as in transporters, enzymes or cell organelles, nitrogen is often considered the most important nutrient for cellular growth. In the following, we will explore the effect of nitrogen and phosphorous on the microalgae Haematococcus pluvialis. An interesting candidate for biotechnological production of astaxanthin, since it can accumulate up to 4% astaxanthin inside the cell. Astaxanthin is a high value carotenoid with beneficial properties for human health. Due to strong antioxidant and anti-inflammatory effects it appears to be a suitable supplement to improve overall health and has even shown positive effects for cancer prevention and treatment.

However, the cell cycle and therefore the growth of H. pluvialis is complex and the differences in morphology during different stages are exceptional (Figure 1). These changes are directly related to the availability of nutrients and can be observed by microscopy, flow cytometry or other visual analysis of the cells.

Figure 1. Life cycle of Haematococcus pluvialis.

The different cell stages vary from ellipsoidal vegetative cells with two flagella, called zoospores to red spherical cysts, called aplanospores. In addition, there are several transition stages with unique morphological characteristics. In the case of H. pluvialis, the cell cycles are of great importance, because astaxanthin is only formed in red cysts. Despite changes in color and shape, the cells vary considerably in size. While the vegetative zoospores are usually smaller, the red cysts can easily be three times bigger. One reason the cell changes from zoospore to an aplanospore is a lack of available nutrients such as nitrogen or phosphorous. Therefore, a zoospores to aplanospores transition is often observed in batch cultures once nutrients become scarce. Considering a batch cultivation limited only by nutrients we can observe an exponential increase in biomass until a plateau is reached, very similar to bacterial growth. However, unlike bacterial growth, this plateau is not reached once nutrients are fully consumed but only at a later stage. This can be easily explained by looking at the cell cycle. As the cells turn into aplanospores, the internal ratio of nutrients to carbon decreases as the cells continue with the uptake of carbon. The carbon is usually stored in the form of carbohydrates such as starch or in the form of lipids.

Most biological models for bacterial growth use the Monod equation to describe the effect of nutrients or substrate on growth. Hence, one might think that this would also be a viable solution to describe microalgae growth. However, a limitation of Monod based models is that once the dissolved nutrients are depleted, given that the Monod term goes to zero once the nutrients are depleted, it results in zero growth at this point. Although microalgae usually also stop dividing at this point, the biomass concentration still increases due to the increasing size of the individual cells. Thus, a different mathematical description is required. A popular solution is the Droop equation, which considers internal nutrients instead of the dissolved nutrient concentration (Figure 2).

Figure 2 Comparison of the Monod model and the Droop model for biological modelling of microalgae growth.

The simulation above clearly shows that the Droop model is the better choice when calculating microalgal growth in respect to nutrient availability. However, the implementation of this model is more challenging. Since the model considers not only dissolved nutrients but also an internal nutrient quota, additional measurements for model calibration are required. Performing measurements of internal nutrients is significantly more difficult since the nutrients are bound in many different ways (see above) and therefore can´t be measured as easily as dissolved, inorganic nutrient sources such as ammonium or phosphate. Instead, methods are required in which the cells are either completely digested or burned at very high temperatures while supplying enough oxygen.

By Lars Stegemüller