Polymers based on sugars are well-known for their high diversity in chemical composition and secondary structure. The innumerable chemo-diversity based on glycosidic linkage of different sugars, as well as many modifications containing non-sugar constituents (sulfates, organic acid, methyl, amino acids, or amine), leads to a big variety of possible structures (Delattre et al., 2016). The polysaccharides (PS) have diverse structure and architecture based on their type and origin. PS have natural biological functions, starting from energy storage over structural support and cell adherence to protect the organism from various endangerments, e.g., heavy metal toxicity, desiccation, radiation, oxidation, viral infections, and predators (Li et al., 2001; Bazaka et al., 2011; Reichert et al., 2017; Fritzsche et al., 2021; Potnis et al., 2021). The protective properties are mainly found in the secreted PS, known as extracellular polysaccharides (EPS). This sub-category of PS can further be divided into cell-bound polysaccharides (BPS) and released polysaccharides (RPS) (Cruz et al., 2020). Besides EPS, PS are also present inside the cells, known as intracellular polysaccharides (IPS). Due to thickening, gelatinous properties and secondary functions, including emulsification, suspension stabilization, encapsulation, flocculation, film-forming and coating, PS are used in the food and cosmetic industries and also in several other processes where the rheological properties of water have to be altered for easier processing or higher commercial value (Sanford and Baird, 1983; Lewis et al., 2000; Paniagua-Michel et al., 2014; Kumar et al., 2018; Gouda et al., 2022). The biological functions are becoming increasingly interesting for commercial application, particularly in the field of nutraceuticals and therapeutics. Recent discoveries of the antioxidative, immunomodulatory, antitumor, antithrombotic, anticoagulant, anti-inflammatory, antimicrobial, and antiviral activities of PS are of particular interest (de Jesus et al., 2019). Whereas, PS from terrestrial plants, seaweed, fungi and non-phototrophic microorganisms are widely described in the literature, detailed information on chemical structure, rheologic properties, possible industrial relevance (i.e., information regarding process parameters and productivity) of PS obtained from microalgae and cyanobacteria are harder to find (Prybylski et al., 2020), which could be due to lower productivities of phototrophic microorganisms.

EPS from microalgae and cyanobacteria are mainly non-repeating, hetero-polysaccharides with glycosidic bonds to non-carbohydrate components. Pierre et al. (2019) attempted to summarize the available information about the structural deviation of EPS gained from microalgae and cyanobacteria. For phylogenetically closed eukaryotic microalgae, the group concluded some general information about the chemical composition of EPS; however, such statements are less clear for cyanobacteria. The intragenus heterogeneity of EPS composition is highly distinctive (Gaignard et al., 2019; Prybylski et al., 2020) and the chemical diversity is also reflected by the quantity of published biological activity of EPS, occasionally even hypothesizing structure-function relationships.

As the efficacy of these biopolymers is significant (Li et al., 2001; Bazaka et al., 2011; Reichert et al., 2017), the question arises why their commercial application, especially in the field of therapeutics, is not yet realized. One main reason lies in the economic perspective of the production process as the yield of EPS from microalgae and the cost for the upstream and downstream processing are not yet economically been found feasible and are currently the object of research projects, including Spiralg (BBI-H2020) or AnViPi (FNR Germany). The common aspect in these projects is the implantation of EPS production into a product cascade leading to a phase II biorefinery, which describes the concept of making use of side streams and byproducts to gain multiple end products from the biomass. Nevertheless, even if PS serve “only” as an added-value product, the question arises, which process parameters influence productivity or space-time yield to further increase the benefit of the process?

Regardless of whether microalgae and cyanobacteria grow by phototrophic, heterotrophic, or mixotrophic processes, the composition of the culture media has a significant influence on EPS production. Until now, the main focus of investigations was on the supply of the elements N, P, and S. It was found that starvation of these three nutritional components led to a higher expression of EPS into the supernatant, while the associated reduction of growth determines the overall productivity. Researchers reported optimal parameters for the supply of carbon with air being enriched with ~2% CO₂ (Iqbal and Zafar, 1993; Cheng et al., 2015).

For phototrophic cultures, light supply is one of the critical process parameters concerning all products from microalgae or cyanobacteria. Generally, there is a positive effect of high light intensity on EPS (Delattre et al., 2016). When comparing different reports, one could conclude that EPS production is enhanced at low irradiations by increasing the light supply, leading to a light inhibition at high light intensities (Trabelsi et al., 2009; Delattre et al., 2016; Silva et al., 2020). The optimum light supply strongly depends on the production organism (Trabelsi et al., 2009; Silva et al., 2020) and the reactor system (Iqbal and Zafar, 1993).

The influence of temperature on productivity is not as easy to describe and is highly species- and strain-dependent. Notably, optimum temperature for EPS production does not always overlap with the optimum culture growth temperature. Botryococcus braunii is described as being highly dependent on a narrow temperature window in which, both fast growth and high EPS production, can be achieved (Iqbal and Zafar, 1993). For Anabaena sp., it was observed that a sudden increase in temperature from 30 to 35°C (optimum growth) to 40–45°C increased (4–5-fold) the release of EPS. Similar behavior is described for Dictyosphaerium chlorelloides, where an increase of 5°C decreases the growth by 30% but doubles the EPS production (Kumar et al., 2017). Other very relevant studies regarding the influence of light and temperature on EPS production for photoautotrophic growing microorganisms have been published. An overview of some outcomes is provided in Table 1.

In this study, the prokaryotic microalga Arthrospira platensis and the eukaryotic unicellular green alga Chlamydomonas asymmetrica were used to identify the influence of process parameters and operational techniques on the formation and productivity of EPS, hoping to find some intragenus trends by investigating to microorganism from different domains.

Arthrospira platensis is a well-known and studied cyanobacteria, limited information has been published about C. asymmetrica, a recent study by group of researchers shows its prospect as a lutein production organism, reporting lutein concentrations of up to 30 mg per gram dry biomass (Hahm et al., 2021).

The effects of light and temperature on EPS production were evaluated in two different phototrophic organisms, the cyanobacterium A. platensis, and the green alga C. asymmetrica. Both process parameters influenced the amount of produced EPS and differ for both organisms. To estimate the comparability of different parameter settings regarding EPS production, we fitted the results of the growth behavior with the growth model of Jung et al. (2021). The significant fit of the model to the experimental data, using the same kinetic parameters (KS, μ_max) for all temperature and light intensity variations, indicates a low bias to other influencing factors. When evaluating all experiments by comparing the experimental biomass development of the cultures with the growth model, a significance level of P < 0.005 is estimated for the experiments with A. platensis and C. asymmetrica, by using the Chi-square test (χ² = 1.2, n = 63) The residuals between the model and the experimental data are independent and well-distributed. Regarding the growth behavior of A. platensis, in this study the evaluated growth parameter K_S (61,2 μmol m⁻² s⁻¹) is similar to those published by Levert and Xia (2001) and Cornet and Dussap (2009) with around 80 μmol m⁻² s⁻¹. Using a K_S value of 80 μmol m⁻² s⁻¹ for fitting our data (μ_max remain unchanged) leads to insignificant changes of the quality of the fit. For example, it was observed that an increase of the quality of the fit by 5 percent points (average relative error) for the culture 975 μmol m⁻² s⁻¹ and 35°C. Fitting the model to the experimental data for culture growth at 25°C and 277 μmol m⁻² s⁻¹ lead to a 3 percent point decrease of the average relative error. The maximum growth rate at 35°C is estimated by the model to μ_max = 2.12 d⁻¹, which is in agreement with the values given from Huang and Chen, who reported global growth rate of μ_max = 2.0 d⁻¹ (Huang and Chen, 1986), and the growth rate published by Lee et al. with μ_max = 2.06 d⁻¹ (Lee et al., 1987). The standard textbook for A. platensis cultivations from Vonshak states the optimum growth temperature window between 35 and 38°C (Vonshak, 1997), which is congruent to the data of current study. In the current study, the growth reduced by 42% comparing the highest and the lowest growth rate, the same reduction in growth by temperature is reported by De Oliveira et al. (1999), investigating a temperature range (20–35°C).

In the case of C. asymmetrica, such a comparison with literature data is strongly biased, as the only published growth parameters regarding this green alga originates from the authors of this study. However, regarding an evaluated culture growth of μ≈2.5 d⁻¹ at optimum conditions, a light-dependent growth being describable by Monod kinetics with an optimal growth temperature of 30°C is unexceptional and lies within an expectancy range (Lee et al., 2015). Thus, the goodness-of-fit of the model and the comparability of the current findings and growth data to published data leads to the conclusion that the growth behaviors and culture developments observed in these experiments are plausible.

Regarding the EPS production of both investigated microalgae, it should be stated that C. asymmetrica achieves 10-fold higher EPS concentrations than A. platensis (up to c_EPS = 1.4 g L⁻¹), yields (up to Y_EPS/X = 1.12 g g⁻¹) and space-time yields (up to STY_EPS = 0.84 g L⁻¹ d⁻¹). These high values for EPS production are similar to those described for Phorphyridium sp. with concentrations c_EPS> 1 g L⁻¹ (Singh et al., 2000) but are even surpassed by the EPS production of Botryococcus braunii, which is reported with c_EPS = 4.5 g L⁻¹ (Lupi et al., 1991). Comparing EPS data with literature can be error-prone, as the culture conditions or age at harvesting are often not clearly stated. The measurable carbohydrates in the supernatant increase with cell breakage, especially noticeable with prolonged retention in the stationary phase. Margo et al. reported for A. platensis a 2-fold rise in EPS concentration compared to early stationary phase (c_EPS = 0.055 g L⁻¹ and death phase (c_EPS = 0.108 g L⁻¹) (Magro et al., 2018). The data from the early phase, as well as the EPS concentrations published by Strieth et al. (Strieth et al., 2020), are a similar range as the concentrations in current experiments (0.012–0.138 g L⁻¹) reported here.

Although the effect of temperature on EPS production is known to be inconsistent between different organisms (Delattre et al., 2016), in current experiments, increasing temperature had a positive influence on EPS production by A. platensis and C. asymmetrica. In a study about exopolysaccharides from cyanobacteria, Nicolaus et al. (1999) described for A. platensis that in the absence of NaCl the EPS production slightly decreased with increasing temperatures, which is contradictory to the current findings and those of Trabelsi et al. (2009) and Lee et al. (2012). This may lead to the conclusion that the absence of NaCl was the primary influencing factor. Trabelsi et al. and Lee et al. investigated the influence of light and temperature on EPS production. Both publications show increasing EPS release at higher cultivation temperatures. The highest concentration reported by Trabelsi et al. is c_EPS = 0.297 g L⁻¹ at 35 °C and 180 μmol m⁻² s⁻¹, whereas Lee et. al reached c_EPS = 0.141 g L⁻¹ at 38°C and 36 μmol m⁻² s⁻¹ (Trabelsi et al., 2009; Lee et al., 2012). Both findings are comparable to the current study of EPS concentrations of c_EPS = 0.138 g L⁻¹ at 35°C and 180 μmol m⁻² s⁻¹. The differences in the total amount of EPS may originate in the different harvesting times. Our experimental setup was designed to harvest at the early stationary phase, not to be biased by the release of IPS due to cell lysis. In contrast, the previous report used data form the late stationary phase. The comparison between growth and EPS concentration published by Trabelsi et al. perfectly shows the accelerated increase of EPS concentration after reaching the stationary phase, where biomass starts declining (Trabelsi et al., 2009). Studies on the temperature dependency of the cyanobacterium Cyanobacterium aponinum showed that the highest EPS productivity was at 35°C (STY_EPS~9 mg L⁻¹ d⁻¹), whereas the highest growth rate was at 40°C. Although, the productivity of A. platensis is about twice as high (STY_EPS = 24 mg L⁻¹ d⁻¹) at similar culture conditions (76 μmol m⁻² s⁻¹, 35°C), the observed trend of reaching the highest EPS productivity at slightly lower temperatures than the optimum growth is similar (Gris et al., 2017).

In the well-researched review by Delattre et al. a general rule-of-thumb was formulated: higher light intensities lead to higher EPS release (Delattre et al., 2016). Results of current study regarding the influence of light on EPS production by C. asymmetrica are supporting this statement. In both experimental approaches, the batch and the turbidostat processes with increase of illumination in the range of 96–1,429 μmol m⁻² s⁻¹ enhanced EPS production in terms of concentration, yield, and space-time yield. However, the results obtained for A. platensis showed a different relationship with respect to light than previously described, Trabelsi et al. were able to show a significant increase of EPS-production with stronger illumination in the range between 50 and 180 μmol m⁻² s⁻¹. A similar range was investigated by Lee et al. (2012) by observing a maximum EPS release at 180 μmol m⁻² s⁻¹ with c_EPS = 0.146 g L⁻¹. This is in accordance with the findings of Dejsungkranont et al., who proposed an optimum light intensity of 203 μmol m⁻² s⁻¹ for EPS production (Dejsungkranont et al., 2017). Those results support our findings that the optimum light intensity is around 100–200 μmol m⁻² s⁻¹ with maximum EPS concentrations of c_EPS = 0.138 g L⁻¹ The significant decrease in EPS production by increasing irradiation in the range of 180–975 μmol m⁻² s⁻¹ correlates with the findings of Silva et al., who reported a reduction in EPS yield from Y_EPS/X = 0.12 g g⁻¹ at 200 μmol m⁻² s⁻¹ to Y_EPS/X = 0.054 g g⁻¹ at 1,000 μmol m⁻² s⁻¹ (Silva et al., 2020). In different experiments of current study, the specific EPS concentration dropped from Y_EPS/X = 0.106 g g⁻¹ at 180 μmol m⁻² s⁻¹ to Y_EPS/X = 0.026 g g⁻¹ at 975 μmol m⁻² s⁻¹. It should be mentioned here that the highest specific EPS concentration was reached at 74 μmol m⁻² s⁻¹ with Y_EPS/X = 0.166 g g⁻¹.

Interestingly, the experiments in the continuous processes showed a lower light influence on EPS concentration and yield than in the results of the batch experiments, with a slight positive effect on the space-time yield, which can be explained by the higher dilution rates. Comparing the two process modes, the results of current study indicate that higher EPS concentrations can be achieved compared to continuous systems. The cultivation of A. platensis at higher light intensities (above 180 μmol m⁻² s⁻¹), is an exception, since the continuous system performed better under these conditions. Nevertheless, this strongly resembles the Gaden type III category of processes, traditionally operated in batch mode (Gaden, 2000).

For A. platensis, the light intensity with the highest EPS release was found to be 180 μmol m⁻² s⁻¹. Stronger irradiations resulted in a substantial decrease in EPS production. The experiments regarding the temperature influences, conducted at a light intensity of 277 μmol m⁻² s⁻¹, suggested an optimum for EPS production at 38°C. However, the best combination of process conditions was found to be 180 μmol m⁻² s⁻¹ and 35°C with c_EPS = 0.138 g L⁻¹. The optimum parameter combination for C. asymmetrica, according to our results, is 30°C and 1,429 μmol m⁻² s⁻¹ where an EPS concentration of cEPS=1.2 g L-1 was reached. Combining the high EPS concentrations with the fast growth, C. asymmetrica is the better production organism, reaching 30 times the space-time yield of A. platensis with STY_EPS = 0.86 g L⁻¹ d⁻¹. Although the turbidostat process with C. asymmetrica led to the highest space-time yield in our experiments, continuous systems are generally not suitable for high production rates—especially at low biomass concentrations.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

S-HJ, NZ, FB, CM, and CL planned and designed the research and interpreted the results. S-HJ, NZ, and FB performed the experiments and proceeding of the raw data. S-HJ and CL wrote the manuscript. S-HJ, NZ, FB, UT, CR, CM, and CL revised the manuscript. All authors read and approved the final manuscript.

This work was funded by the German Federal Ministry for Education and Research (BMBF 03SF0457) and the German Federal Ministry of Food and Agriculture (FNR 22017518 and 2219NR287).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.