Characteristics of the growth rate and lipid production in fourteen strains of Baltic green microalgae

All strains showed a rapid increase in the number of cells during the 21-day period of culture (Table 2). There was no case of growth inhibition or decline in the number of cells. Strains belonging to the Chlorella genus exhibited the highest growth rate, ranging from 0.39 d^-1 to 0.5 d^-1, and thus the highest final cell number (up to 3.72 × 10⁷ cells ml^-1). The results correspond to growth rates obtained for other Chlorella strains, e.g. Kong et al. (2011) reported the growth rate at the level of 0.32 d^-1 in the phototrophic culture. Remaining strains exhibited a lower growth rate, varying within the range of 0.27 d^-1 and 0.38 d^-1. Differences observed in species-specific growth rates may be explained by the fact that the growth of unicellular microalgae is inversely proportional to their cell size (Banse 1976). This was also observed in the present study. The strains with the highest cell volume, e.g. Scenedesmus sp. BA-147, showed a rather low growth rate and thus a lower final cell count compared to small-sized species, e.g. Chlorella minutussima BA-12, the volume of which was ca. 20 times smaller than Scenedesmus sp. BA-147 (Tables 1, 2). This is due to the fact that larger cells exhaust metabolic energy more quickly, which results in slower growth (Borowitzka 1992). The size of a cell also determines its surface area to volume ratio (SA:V ratio). This consequently affects the efficiency of nutrient uptake by cells; small cells with the higher SA:V ratio assimilate nutrients more effectively (Fogg 1975). The opposite effect is observed in larger cells, where also the supply of various substances from the cell surface to its interior through diffusion is less effective (Nielsen 2006). Consequently, size-dependent nutritional properties of microalgae regulate their overall metabolism and hence their growth rate (Fogg 1975; Hein et al. 1995).

Table 2

Growth and lipid accumulation characteristics in the tested strains

Furthermore, it was also proved that there are size-dependent maximal densities of cells in microalgal cultures, regardless of their type (Agustí et al. 1987). The study showed that algae with larger cells grew more slowly and maintained lower biomass due to the extinction of light caused by the cells themselves. This phenomenon, called self-shading, was by far the most important limiting factor. The growth form of microalgae also affects their growth rates and thus colony-forming species with larger cells may reach densities close to those observed in smaller species. In the set of tested microalgae, the strain of colonial Dictyosphaerium sp. BA-157 grew well, reaching high final cell density of 9.84 × 10⁶ cells ml^-1 and being among the most abundant strains of similar size (Table 2). The research showed that there is no clear relationship for the size-dependent maximum growth of microalgae growing in colonies, because they are a form of evolutionary adaptation that allows avoiding constraints resulting from increasing cell size (Nielsen 2006).

Rapid growth rates are considered an important feature of microalgal species cultured for commercial purposes, providing a competitive edge over microorganisms (including other algal species) that cause contamination of outdoor cultures. Small-sized fast-growing microalgae also require less culture space due to the higher cell density (Tan & Lee 2016). Whereas considering the forms of algal growth, species exhibiting the colonial growth are able to reduce grazing pressure (Nielsen 2006).

Significantly higher cellular lipid content was observed in 4 strains with the highest cell volume, i.e. O. submarina BA-01 (16.19 pg cell^-1), Scenedesmus sp. BA-147 (24.81 pg cell^-1), O. cf. submarina BA-172 (15.54 pg cell^-1) and Microthamnion sp. BA-173 (12.23 pg cell^-1) (p < 0.05, Fisher LSD test), while in other strains it ranged from 1.68 pg cell^-1 in C. minutussima BA-12 to 6.26 pg cell^-1 for Monoraphidium sp. BA-165. The cellular lipid content patterns among the strains were not reflected in the final lipid yield values as they also depend on the growth rate of the strain. In the 21^st day of culture, lipid yields varied within the range of 3.50 mg l^-1 (Chlorella vulgaris BA-76) and 81.13 mg l^-1 (Monoraphidium sp. BA-165). The comparison of values obtained for all tested strains enabled the identification of cultures with significantly higher final lipid yields, i.e. C. minutissima BA-12 (62.52 mg l^-1), C. fusca BA-18 (64.85 mg l^-1), C. vulgaris BA-80 (75.90 mg l^-1), Chlorella sp. BA-167 (80.53 mg l^-1) and Monoraphidium sp. BA-165 (81.13 mg l^-1) (p < 0.05, Fisher LSD test), which, as mentioned earlier, were also characterized by high growth rates and final cell densities (Table 2).

Griffiths & Harrison (2009) showed the volumetric lipid productivity (P_LV) as a universal parameter and a key characteristic when choosing algal species for valuable biomass production. It combines the growth rate of strains (calculated for the exponential growth phase) and volumetric lipid yield, giving easily comparable results among various microalgae strains. Fig. 1 presents productivity rates for all tested strains. Among them, five strains belonging to the Chlorella genus, namely BA-02, BA-12, BA-18, BA-80, BA-167, and Monoraphidium strain BA-165 showed significantly higher productivity values, varying within the range of 23.86 and 37.42 mg l^-1 d^-1 (Fisher LSD test; p < 0.05). The C. vulgaris BA-80 strain was marked with the top lipid productivity of 37.42 mg l^-1 d^-1 and high growth rate (0.49 d^-1). In the second most productive strain, i.e. Chlorella sp. BA-167, the growth rate was lower (0.39 d^-1), resulting in lower lipid productivity.

Figure 1

Although the volumetric lipid productivity is a very useful parameter in assessing the potential of microalgal strains for the mass culture and lipid production, it is advisable to compare the calculated values with other parameters such as the lipid yield after a certain period of culture, but before the full stationary phase (Wood et al. 2005; Borowitzka 2013; Xu & Boeing 2014). In this study, both parameters indicated the five strains as the most promising ones for biomass production, i.e. C. minutussima BA-12, C. fusca BA-18, C. vulgaris BA-80, Monoraphidium sp. BA-165 and Chlorella sp. BA-167.

The range of lipid productivity recorded in the literature covers values that differ by several orders of magnitude (Table 3), reaching up to 200 mg l^-1 d^-1 (Nascimento et al. 2013). There are severalfold differences among various microalgal species and strains grown in laboratory batch cultures. For instance, the green alga Botryococcus braunii, often marked with the highest lipid yields, was characterized by considerable variations with respect to its growth rate and productivity (5.51 vs. 112.43 mg l^-1 d^-1), resulting from the B. braunii strain-specific characteristics, the physiological state of strains as well as culture conditions (Metzger & Largeau 2005; Tasi et al. 2016). Productivity of most of the selected Baltic strains tested in this study fits well within the range of P_LV reported in the literature (Table 3). Taking into account the tested strains of the Chlorella genus, it was shown that for some of them, i.e. BA-2, BA-12, BA-80 and BA-167, the lipid productivity rates were similar to those observed in other research screening microalgal strains for most efficient producers of lipids (Table 3). For instance, Lee et al. (2010) recorded Chlorella strain productivity at the level of 11.1 mg l^-1 d^-1, although at slightly higher light intensity, i.e. 150 µmol m^-2s^-1, and aeration of the culture medium. When culturing C. minutussima, Illman et al. (2000) also recorded the production of lipids around 10 mg l^-1 d^-1, but at the lower light intensity (77 μmol m^-2s^-1) and with considerably higher temperature (25°C). Whereas Griffiths et al. (2012) determined almost three times higher productivity rate, 27 mg l^-1 d^-1, for C. vulgaris grown at 25°C and light intensity of 250 µmol m² s^-1 in the medium enriched with CO₂. In this study, the P_LV values estimated for the most productive Chlorella strains under relatively lower temperature (compared to other screening studies for high lipid content strains) were around 30 (up to 37) mg l^-1 d^-1, falling within the upper range of microalgal lipid productivity compared to literature data (see Table 3). The presence of Monoraphidium sp. BA-165 among the strains with the highest P_LV values is also not surprising as previous studies have already confirmed the potential of this genus for increased production of lipids (Yu et al. 2012; Bogen et al. 2013).

Table 3

Volumetric lipid productivity (PLV) of green algae reported in literature, listed together with Baltic strains indicated in this study as characterized by high productivity

To identify the most promising strains for mass culture, cluster analysis based on growth rates, final cell density and lipid yields was carried out. The tested strains were grouped into two main groups, each of which was then divided into two specific branches, indicating strains with similar characteristics (Fig. 2). The upper branch grouped species tentatively described as those having low potential for efficient mass culture to produce lipids; the strains were characterized by low to moderate growth rates and yields of lipids (Table 2). Whereas the second group contained strains that could be defined as very promising for the production of lipids. Within this group, two specific clusters were distinguished: one containing two strains, Monoraphidium sp. BA-165 and Chlorella sp. BA-167, revealing the highest lipid yield and the second one containing four strains, C. vulgaris BA-02, C. minutussima BA-12, C. fusca BA-18 and C. vulgaris BA-80, which were characterized by the highest values of the volumetric lipid productivity, i.e. the highest growth rate and only slightly lower lipid yield (Table 2). In this study, temperature, light intensity and salinity conditions were set to enable the uninhibited growth of microalgae, but not necessarily to obtain their highest possible efficiency. Therefore, there is still plenty of scope for improvement and stimulation of productivity to reach much higher lipid yield values, e.g. higher temperature, stronger light, CO₂ enrichment or stress factors such as nitrogen limitation or salinity stress (Courchesne et al. 2009). Referring to Table 3, a significant increase in microalgal productivity was observed with CO₂-enriched aeration and at higher temperature, indicating their possible influence. This confirms the need for further research on the conditions of lipid productivity stimulation, affecting local strains, because algal strains of the same species have different biochemical profiles and stress responses, depending on the natural habitat conditions and phenotype plasticity in a single genotype (Miner et al. 2005). A recent study confirmed that brackish water species have slightly higher lipid content under stress compared to the typical marine species, which makes Baltic strains even more promising in biotechnological applications (Schwenk et al. 2013).

Figure 2