Cultures of microalgae gain increasingly more attention due to their high biomass productivity, which fits perfectly into the global trend of seeking new renewable energy and other desirable high value product sources (Spolaore et al. 2006). Rapid growth, the ability to accumulate large amounts of pigments, carbohydrates and lipids in cells are preferred features in the production of biofuels or natural bioactive compounds (Patterson et al. 1994). In addition, new materials offer many benefits for the environment, such as more efficient land use compared to terrestrial plants, CO2 sequestration as well as the possibility of wastewater cleaning. Therefore, culturing microalgae on a larger scale can provide both economic and environmental benefits.
Large-scale production, in order to be profitable and undisturbed by external factors, requires proper optimization, which starts with the selection of suitable species and determination of their optimal growth conditions (Spolaore et al. 2006; Mata et al. 2010; Lam & Lee 2012; Tsarenko et al. 2016). It has been noted that rigorous selection for biotechnological use is challenging due to varying sets of characteristics between strains i.e. growth rate, cell size and the content of metabolism products (Griffiths & Harrison 2009). Depending on the purpose of mass culture and due to the fact that there are no strains satisfying all the demands, strains with a proper set of characteristics are selected. There are already strains indicated as having high potential to be used in mass cultures, i.e. species belonging to
Recently, more emphasis has been put on the content of high-energy compounds needed to increase the energy of biomass obtained from algae. Microalgae produce lipids as a storage material in the form of triglycerides (TAGs) or more complex lipids performing specific functions in the cell membranes (Khozin-Goldberg 2016). Studies have shown that the high cellular content of the desired product, such as triglycerides, in the case of biodiesel is not sufficient itself and must be accompanied by an adequate growth rate. In this respect, the algae to be selected should have a high growth rate and high lipid content during unstressed (exponential) growth (Borowitzka 2013).
The optimal growth conditions for several Baltic strains have been sufficiently described in the literature (e.g. Latała et al. 2006), although studies on their potential use in biotechnology are still in the initial phase. Culturing Baltic microalgae may be preferred because of their high adaptability to a relatively wide range of environmental conditions occurring in the Baltic Sea, e.g. relatively low salinity (8 PSU) and a wide range of temperature changes (0–2°C in winter and up to 22°C in summer). Recent screening tests of microalgae for valuable biomass production have shown that brackish Baltic strains performed equally well or even better in terms of biomass quality and yield compared to other strains, often used in algal research and development (Olofsson et al. 2015).
Therefore, this paper aims at comparing the growth rate and lipid production in fourteen selected strains of Baltic green microalgae. The strains were incubated under non-stressed conditions and their final cell count, growth rate as well as lipid yield and productivity were examined in order to identify those with inherent features of outstanding lipid production.
The study included fourteen strains of green algae maintained as monocultures in the Culture Collection of Baltic Algae (CCBA; Latała et al. 2006) located at the Institute of Oceanography, University of Gdańsk (Table 1).
List of strains used in the study and their morphological characteristics. Species names are consistent with the catalog of the CCBA collection.
Strain
CCBA designation
Cell volume min.- (median)-max [μm3]
Cell shape
BA-1
42–(92)–285
rotational ellipsoid
BA-2
65–(133)–303
sphere
BA-3
45–(147)–260
rotational ellipsoid
BA-12
8–(18)–73
sphere
BA-18
33–(113)–2144
sphere
BA-76
15–(35)–103
rotational ellipsoid
BA-80
65–(155)–425
sphere
BA-147
66–(244)–707
rotational ellipsoid
BA-157
31–(131)–298
sphere
BA-165
34–(67)–120
2 cones
BA-167
19–(50)–126
sphere
BA-168
5–(12)–24
cylinder
BA-172
66–(129)–388
rotational ellipsoid
BA-173
48–(131)–237
cylinder; filamentous
Microalgae were grown in 100 ml glass Erlenmeyer flasks filled with 50 ml of f/2 medium (Guillard 1975) prepared on the basis of artificial sea water (
The volume of cells was calculated according to the instruction provided in Olenina et al. (2006).
The specific growth rates (μ; d-1) of the tested strains were determined by counting cells using Bürker haemocytometer and calculated according to the equation provided by Guillard (1973):
where
Quantitative measurements of lipids were performed using the optimized colorimetric sulfo-phospho-vanillin method (SPV method) by Chabrol & Charonnat (1937). To extract lipids, 1 ml aliquots of algal cultures were centrifuged (10 000 rpm, 5 min) and re-suspended in 0.5 ml of methanol. To fully disrupt the cell wall and to improve the extraction of lipids, glass beads were added and then samples were vigorously shaken for 10 min at 2000 rpm. Subsequently, 1 ml of chloroform was added and the shaking procedure was repeated (Folch et al. 1957). Then, samples were centrifuged, supernatant was collected and supplemented with 0.2 ml of 0.8% NaCl solution. Samples were then left in room temperature until two layers were formed and the upper layer was collected and discarded. The lower layer was dried under N2 at 50°C. Next, 0.3 ml of concentrated sulfuric acid was added and samples were heated at 90°C for 10 min. After that step, 1 ml of SPV reagent (1.2 g vanillin per 1 liter of 68% phosphoric acid) was added in order to fully stain the fatty acids and the samples were incubated at 36°C for 5 min. The absorbance of the final solution was measured at 525 nm (Knight et al. 1972). The concentration of lipids was estimated based on the calibration curve that was developed using high-quality soybean oil as a source of fatty acids, which has a similar composition of lipids compared to algae (Grama et al. 2014).
The volumetric lipid productivity values of the examined strains were calculated according to the equations recommended by Griffiths & Harrison (2009) and Xu & Boeing (2014):
where PLV is the volumetric lipid productivity [mg l-1 d-1], μ is the specific growth rate [d-1] and
To compare the mean values of the analyzed parameters, analysis of variance (ANOVA) was performed (Stanisz 2007a). In order to separate the strains with the highest potential for lipid production, cluster analysis was employed, using the agglomerative hierarchical clustering method based on the final cell density, growth rates and lipid yields (Stanisz 2007b). All statistical analyses were performed using Statistica 10 (StatSoft Inc., USA).
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
Growth and lipid accumulation characteristics in the tested strains
Strain
Final cell count [cells ml−1]
Growth rate μ [d−1]
Lipid yield [mg l−1]
Cellular lipid content [pg cell−1]
3.56 × 106 ± 7.72 × 104
0.35 ± 0.01
57.59 ± 0.50
16.19 ± 0.29
1.13 × 107±2.66 × 105
0.42 ± 0.01
56.82 ± 6.29
5.05 ± 0.43
7.25 × 106 ±1.53 × 105
0.38 ± 0.01
38.64 ± 2.17
5.33 ± 0.33
3.72 × 107 ± 7.25 × 106
0.5 ± 0.06
62.52 ± 9.78
1.68 ± 0.26
1.64 × 107 ± 3.85 × 105
0.45 ± 0.01
64.85 ± 10.81
3.96 ± 0.64
1.33 × 106 ± 7.83 × 105
0.26 ± 0.03
3.50 ± 3.13
2.63 ± 2.51
3.07 × 107 ±1.44 × 106
0.49 ± 0.01
75.90 ± 9.32
2.48 ± 0.41
1.87 × 106 ±2.02 × 105
0.26 ± 0.01
46.39 ± 8.49
24.81 ± 2.70
9.84 × 106 ± 3.71 × 105
0.30 ± 0.01
31.57 ± 3.44
3.21 ±0.15
1.30 × 107 ± 3.65 × 105
0.35 ± 0.01
81.13 ± 15.91
6.26 ± 1.29
1.65 × 107 ± 2.64 × 105
0.39 ± 0.01
80.53 ± 3.60
4.89 ±0.13
1.95 × 107 ± 3.38 × 106
0.35 ± 0.01
16.80 ± 4.34
0.86 ±0.15
2.80 × 106 ± 9.05 × 104
0.24 ± 0.01
43.47 ± 7.29
15.54 ± 2.90
4.61 × 106 ±1.70 × 105
0.30 ± 0.01
56.41 ± 2.74
12.23 ± 0.14
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
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.
Griffiths & Harrison (2009) showed the volumetric lipid productivity (PLV) 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
Volumetric lipid productivity (PLV) in the tested Baltic green algae strainsFigure 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.
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
Volumetric lipid productivity (PLV) of green algae reported in literature, listed together with Baltic strains indicated in this study as characterized by high productivity
Species/strain
PLV [mg × l−1 × day−1]
Culture conditions temp.; light; medium
References
5.51
25°C; 150 μmol m2 s−1; CHU13
(Yoo et al. 2010)
6.88
25°C; 70 μmol m2 s−1; BG11
(Yu et al. 2012)
6.91
25°C; 150 μmol m2 s−1; BG11
(Yoo et al. 2010)
9.50
150 μmol m2 s−1
(Lee et al. 2010)
10.24 (calculated)
25°C; 77 μmol m2 s−1; Guillard’s marine medium
(Illman et al. 2000)
11.10
150 μmol m2 s−1; BG11
(Lee et al. 2010)
11.30
28°C; 54 μmol m2 s−1; BG11
(Harwati et al. 2012)
11.50
150 μmol m2 s−1; BG11
(Lee et al. 2010)
17.23
18°C; 100 μmol m2 s−1; f/2
this study
19.92
18°C; 100 μmol m2 s−1; f/2
this study
20.65
25°C; 140 μmol m2 s−1; CHU13; CO2 enriched
(Yoo et al. 2010)
23.89
18°C; 100 μmol m2 s−1; f/2
this study
24.22
25°C; 140 μmol m2 s−1; CHU13; CO2 enriched
(Nascimento et al. 2013)
26.77
25°C; 140 μmol m2 s−1; CHU13; CO2 enriched
(Nascimento et al. 2013)
27.00
25°C; 250 μmol m2 s−1; 3N BBM; CO2 enriched
(Griffiths et al. 2012)
28.85
18°C; 100 μmol m2 s−1; f/2
this study
29.00
25°C; 250 μmol m2 s−1; 3N BBM; CO2 enriched
(Griffiths et al. 2012)
29.49
18°C; 100 μmol m2 s−1; f/2
this study
31.23
18°C; 100 μmol m2 s−1; f/2
this study
31.26
18°C; 100 μmol m2 s−1; f/2
this study
37.43
18°C; 100 μmol m2 s−1; f/2
this study
56.07
25°C; 140 μmol m2 s−1; CHU13; CO2 enriched
(Nascimento et al. 2013)
112.43
25°C; 140 μmol m2 s−1; CHU13; CO2 enriched
(Nascimento et al. 2013)
204.91
25°C; 140 μmol m2 s−1; CHU13; CO2 enriched
(Nascimento et al. 2013)
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,
Cluster analysis of the tested Baltic green algae strains based on their growth rates and lipid yield dataFigure 2
Fourteen selected Baltic green algae strains manifested different growth rates with a clear effect of cell size. Strains belonging to the