Evaluation of Arthrospira platensis growth under different cultivation conditions
The growth curves of A. platensis cultivated in different culture conditions are presented in Fig. 1. As observed, none of the conditions presented an adaptation phase. Condition 6 resulted in the highest growth rate (µ=0.55), in medium with 2 g.L-1 of NaNO3 and under 600 μE.m-2.s-1 of PFD. Condition 2 led to the lowest growth rate, with 1.125 g.L-1 of NaNO3 and a PFD of 200 μE.m-2.s-1, followed closely by number 1 (0.25 g.L-1 of NaNO3 and 200 μE.m-2.s-1 of PFD) and condition 3 (2 g.L-1 of NaNO3 and 200 μE.m-2.s-1 of PFD). All three of these conditions corresponded to the lowest PFD used. The results demonstrate that the A. platensis growth rate was practically independent of the NaNO3 concentration, and indicates that the PFD was the limiting factor.
The PFD values may have affected the growth rate, which increased until reaching a point of light saturation and, from this point, there may even have been a decrease in the rate of growth as a consequence of photo inhibition (Carvalho et al. 2011). In cultures grown at 1000 μE.m-2.s-1 and with NaNO3 at a concentration of 2 g.L-1, light saturation may have occurred, which resulted in a lower rate of cell growth. In cultures using a PFD at 600 μE.m-2.s-1, the NaNO3 concentration was probably the limiting factor for A. platensis growth.
Final biomass concentration and EPS yield under different growth conditions
Some researchers reported that different concentrations of NaNO3 and PFD could induce the EPS production from cyanobacteria (Aikawa et al. 2012; Dejsungkranont et al. 2017; Ohki et al. 2014; Villay et al. 2013). Modified Zarrouk’s medium has 2.5 g.L-1 of NaNO3. Therefore, for the experimental design, a NaNO3 concentration (2 g.L-1) close to that for which regular cell growth is already known and a concentration reduced by 75% (0.5 g.L-1) were used. For the PFD, the minimum and maximum values that have reportedly been used for A. platensis are 57.2 μE.m-2.s-1 (Chentir et al. 2018), and 700 μE.m-2.s-1 (Aikawa et al. 2012), respectively.
The values of the final biomass concentration and the EPS yield are given in Table 1. In general, the O.D. values were related to the biomass concentration. However, there was some variation for the intermediate values, although the differences between the expected O.D. and the observed O.D. were not so high, suggesting they could have been caused by small measurements error.
Considering the results, the highest final biomass concentration (1.292 g.L-1) was observed under conditions with the highest NaNO3 concentration (2 g.L-1). However, for the EPS yield, the best conditions were achieved under the lowest NaNO3 (0.25 g.L-1) and PFD (200 μE.m-2.s-1), which generated 111 mg.g-1 of EPS. Although the second highest EPS content (100 mg.g-1) was obtained under conditions of the highest values of NaNO3 (2 g.L-1) and PFD (1000 μE.m-2.s-1). In other words, the maximum EPS content (EPS mass/biomass) was attained using both extremes of the experimental design.
The results presented in Fig. 2a and 2b show that the EPS production by A. platensis was not directly associated to its growth. As can be observed, the response in which the highest final biomass concentration was obtained differed from that which generated the highest EPS production. In general, metabolic stress may negatively affect cyanobacteria growth, during which cells use their energy to produce reserve compounds such as carbohydrates and EPS (Santos et al. 2019). Condition 9 was an exception, since this condition promoted one of the highest EPS yields and, at the same time, one of the highest final biomass concentrations.
Table 2 shows the results of the coefficients and their interactions, R2, lack of fit and the p-values for final biomass concentration and EPS yield from A. platensis. A 95% confidence level was adopted (p < 0.05). The model was significant for final biomass concentration with a p-value equal to 0.0379. It was found that the PFD and its interactions have a significant influence on the model. For the EPS production, the model was significant, with a p-value equal to 0.0164, and the variables that showed a significant influence were the PFD and its interactions.
The regression equation was generated, as shown in Eq. 2 and Eq. 3, according to the values presented in Table 2.
Biomass concentration = 1.07 + 0.0755A + 0.0892B + 0.0138AB + 0.0244 A2 – 0.2646 B2 (2)
EPS yield = 44.54 + 8A – 15.67B + 17.75AB + 12.88 A2 + 24.88 B2 (3)
It is noteworthy that the lack of fit values for both biomass and EPS production were significant (p < 0.05). In this case, the model does not have a good predictive capacity. Despite the lack of adjustment, it is possible to visualize the best working regions.
In Figure 2a, the response surface graphics show that the conditions which led to the highest final biomass concentration were a PFD of 600 μE.m-2.s-1 and 2 g.L-1 NaNO3. A decrease in biomass production was also observed when the value of PFD was smaller. The PFD can influence cyanobacteria photosynthesis. Excessive or insufficient light may affect the biomass productivity and yield of metabolic products (Carvalho et al. 2011). It is likely that the nitrogen-reduced conditions in this work did not compromise the biomass production and were high enough to sustain the cell growth. However, the lowest PFD negatively affected the biomass production. In this case, it seems that light intensity was insufficient to support the biomass production. Aikawa et al. (2012) also observed that biomass production was related to the PFD, and that A. platensis produced the highest final biomass concentration (1.6 g.L-1) at 700 μE.m-2.s-1, the highest PFD value they tested. Under the smallest PFD value, 20 μE.m-2.s-1, a much smaller final biomass concentration (0.1 g.L-1) was attained.
Figure 2b shows the response surface obtained for the EPS content (mg.g-1). The highest values were observed at the two extremes, 111 and 100 mg.g-1, using conditions 1 and 9, respectively, in addition to condition 3, which also generated an EPS yield of 100 mg.g-1. The EPS production from A. platensis was also affected by the PFD; however, it was not the only determinant variable. NaNO3 starvation contributes to the increase in the C/N ratio. Consequently, it promotes the incorporation of carbon reserve in the EPS (Otero and Vincenzini 2003). The lowest PFD (200 μE.m-2.s-1) and NaNO3 concentration (0.5 g.L-1) resulted in the highest EPS yield. It is possible that the combined effects led to cells producing more EPS in response to the limited conditions. This result is in accordance with that found by Chentir et al. (2018), who obtained the highest EPS yield, 0.902 g.g-1, using 0.5 g.L-1 NaNO3 combined with a PFD of 57.2 μE.m-2.s-1 in A. platensis.
Chentir et al. (2017) evaluated the maximization of EPS production from A. platensis as a function of variations in NaCl concentration and PFD. Although the PFD did not have any positive effect on EPS production, its interaction with the NaCl concentration provided a 0.98 g.g-1 yield of EPS. On the other hand, Dejsungkranont et al. (2017), through studying the effect of PFD on the production of EPS from A. platensis, observed that the highest level of PFD (203 μE.m-2.s-1) favored EPS production (956.4 ± 37.3 mg.L-1) as well as biomass (1.5 g.L-1). Whereas under the lowest PFD (101 μE.m-2.s-1), 0.8 g.L-1 of biomass and 637.3 ± 41.3 mg.L-1 of EPS were obtained.
Trabelsi et al. (2009) carried out a study evaluating the effect of different temperatures and PFD on the final biomass and EPS concentrations by A. platensis, and described a possible correlation between the two responses. According to the authors, the production of biomass and EPS are mutually dependent, and the increase of EPS production may be associated with the kinetics of growth. To achieve a greater EPS content, it is necessary to optimize the PFD while the temperature should be maintained between 30 and 35ºC. In that work, A. platensis produced the maximum EPS content at the highest PFD used, 180 μE.m-2.s-1, with 297.4 ± 11.1 mg.L-1. The result for EPS content found by Trabelsi et al. (2009), 210 mg.L-1, under 100 μE.m-2.s-1 and 2.5 g.L-1 NaNO3, was twice that of our result, 91 mg.L-1.
The results found in the present study demonstrate that the choice of the best culture conditions depended on the response of interest. The results in Figures 2a and 2b showed that the production of EPS by A. platensis was not directly associated to growth. Nevertheless, it is possible to choose a condition in which the EPS content as well as biomass concentration could be produced at reasonable values.
Other studies in the literature using different microorganisms have reported the effect of culture conditions on the EPS content and biomass concentration. For Cyanothece sp. 113, NaNO3 concentrations between 0 - 200 mg.L-1 were used (Su et al. 2007). In this case, the final biomass concentration was observed to increase, reaching 1.2 g.L-1 with a NaNO3 concentration of 74.3 mg.L-1, but then decreased at values higher than 100 mg.L-1. However, a decrease in EPS concentration, from 7 g.L-1 to 5g.L-1, was reported elsewhere when 200 mg.L-1 of NaNO3 was used. In the same work, the PFD effect was evaluated in the 20-100 μE.m-2.s-1 range and the best condition was found to be 86 μE.m-2.s-1 for both biomass and EPS production (Su et al. 2007).
FTIR analyses
Figure 3 shows the FTIR spectrum for EPS samples from A. platensis. The broad bands observed around 3400 cm-1 are attributed to O-H and N-H stretching. The weak absorptions in the region 3000 cm-1 to 2840 cm-1 are associated to C-H asymmetrical and symmetrical stretching modes of methyl and methylene groups. The absorption at 1650 cm-1 is attributed to C=O stretching of carboxylate and amide groups (amide I band). The high-intensity band with maximum at 1442 cm-1 is attributed to more complex vibrations, associated to O-H bending (Can et al. 2019; Trabelsi et al. 2009). The most intense absorption of the spectrum, at 1046 cm-1, may be attributed to C-O-C, S-O and P-O-C stretching vibrations. This result evidenced the presence of polysaccharides, proteins/polypeptides, and of sulphate and phosphate groups linked to polymeric substances.
Thermal analysis
Thermogravimetric analysis is an important technique, as it demonstrates the thermal stability of the EPS (Fig. 4). The EPS displayed two stages of thermal degradation. In the first stage, up to 150ºC, 10% weight was lost and this may be attributed to the loss of water and other volatile substances. In the second stage, the degradation of the polymer chain occurred, between 225 and 350°C, with fifty percent of the total mass lost.
The thermal degradation of EPS from microalgae and cyanobacterium was recently reported. Two stages were observed for the EPS from Dunaliella salina; in the first, 15% weight was lost at up to 150ºC. In the second, 55% of total EPS weight loss was observed with maximum loss at 240°C (Mirsha et al. 2011). On the other hand, for the EPS from Nostoc carneum, three stages of thermal degradation were detected. In the first stage, 15% weight was lost up to 155ºC. In the second stage, with a maximum at 237ºC, 39% weight was lost, attributed to polysaccharide degradation. The third phase occurred up to 378ºC with 32% of weight loss (Hussein et al. 2015).
Carbohydrate and protein quantification
The chemical composition of the EPS depends on environmental conditions and the microorganism studied (Nouha et al. 2018). According to Wingender et al. (1999), the majority of EPS constituents are carbohydrates and proteins. In A. platensis, the carbohydrate and protein content were estimated as 55% and 13%, respectively, in a photoautotrophic growth for 25 days (Pignolet et al. 2013). Carbohydrate and protein contents for A. platensis EPS determined in the present work are shown in Table 3. The highest contents of carbohydrates, 39.5 ± 2.34 mg.g-1 and 38.73 ± 2.55 mg.g-1, were observed under conditions 5 (1.125 gL-1 NaNO3 and 600 μE.m-2.s-1) and 9 (2 gL-1 NaNO3 and 1000 μE.m-2.s-1), respectively.
According to Depraetere et al. (2015), when the nitrate source is depleted, protein synthesis is reduced. In the present work, the highest protein concentrations, 7.05 ±0.30 mg.g-1 and 6.46 ± 0.20 mg.g-1, were observed for intermediate NaNO3 concentrations, under conditions 5 (1.125 gL-1 of NaNO3 and 600 μE.m-2.s-1) and 8 (1.125 gL-1 of NaNO3 and 1000 μE.m-2.s-1), respectively.
Rheological properties
Aqueous solutions of EPS 01 (0.25 gL-1 NaNO3 and 200 μE.m-2.s-1) and EPS 09 (2 gL-1 NaNO3 and 1000 μE.m-2.s-1) were prepared at 5 g.L-1 and 10 g.L-1, and their rheological properties were investigated. Fig. 5a and 5b show the variation of storage modulus, G', and loss modulus, G", with the oscillatory frequency. Their rheological behaviors were quite similar to that previously observed for other polysaccharides, such as gum algaroba, a galactomannan extracted from Prosopis juliflora (Azero and Andrade 2006). At very low frequencies, the viscous character predominated. After the crossing of G' with G", the elastic character was predominant. As expected, the value of the frequency at which the crossing occurs was smaller for the highest concentration. The same behavior was observed for the EPS09 supernatant at 5 and 10 g.L-1.
Our results differ from those reported by Chentir et al. (2017), who also assessed the EPS of A. platensis. The authors described a gel-like behavior, in which the storage modulus values were higher than the loss modulus values at different concentrations (1%, 2.5% and 5%). Furthermore, Mourhin et al. (1993) found a non-Newtonian character for EPS dispersions of Spirulina platensis, which was attributed to its polyanionic nature.