Identification of the newly isolated strains
Sequence comparisons of the ITS2 rDNA revealed the 12 new green algal strains to share high similarities, i.e., 95 -100%, with available references (Table 1). This identified the strains as four different species of Tetradesmus (T. arenicola, T. bajacalifornicus, T. deserticola, and T. obliquus), two species of Desmodesmus (D. armatus and D. multivariabilis), Pseudomuriella aurantica, and Chlorella vulgaris. The strain SAG 2630 shared high sequence similarity (98 %) with an unidentified Chlamydomonas sp. (Volvocales, Chlorophyceae) and, therefore, was left unidentified at the species level.
Table 1
The 12 newly isolated strains, their species identification, origins, sequence accessions, and closest reference sequences.
Strain
|
species identification
|
isolation source; latitude, longitude
|
sequence accession no.
|
length ITS2
|
ITS2 sequence identities with next closest reference
|
sequence accession no. next closest reference
|
SAG 2630
|
Chlamydomonas sp.
|
Temporary freshwater pond with algal bloom (Israel, Haifa); 32.808119 N, 35.020541 E
|
MZ546610
|
234
|
230/235 (98%)
|
MH311547
|
SAG 2629
|
Chlorella vulgaris
|
Temporary freshwater rivulet, iron-rich (Germany, Bad Pyrmont); 51.988757 N, 9.252696 E
|
MZ546604
|
244
|
244/244 (100%)
|
AY591499, and 55 other
|
SAG 2606
|
Chlorella vulgaris
|
Temporary freshwater rivulet, iron-rich (Germany, Bad Pyrmont); 51.988757 N, 9.252696 E
|
MZ546608
|
243
|
244/244 (100%)
|
AY591499, and 55 other
|
SAG 2635
|
Desmodesmus armatus
|
Soil surface of a meadow (Ukraine); 46.480278 N, 33.849722 E
|
MZ546611
|
244
|
245/245 (100%)
|
MK975484, and 12 other
|
SAG 2628
|
Desmodesmus multivariabilis
|
Biofilm on soil surface (Germany, Uslar); 51.649100 N, 9.750686 E
|
MZ546603
|
|
248/248 (100%)
|
MH311545
|
SAG 2631
|
Pseudomuriella aurantiaca
|
Biofilm on soil surface (Israel, Haifa); 32.778451 N, 35.025604 E
|
MZ546609
|
246
|
245/247 (99%)
|
MH703741, and 2 other
|
SAG 2632
|
Tetradesmus arenicola
|
Biological soil crust on the surface of sandy soil (Ukraine); 46.576798 N, 31.512473 E
|
MZ546602
|
239
|
240/240 (100%)
|
MH703775, and 3 other
|
SAG 2633
|
Tetradesmus arenicola
|
Surface of sandy soil (Ukraine)
|
MZ546612
|
239
|
240/240 (100%)
|
MH703775, and 3 other
|
BIOTA 136
|
Tetradesmus bajacalifornicus
|
Biological soil crust on the surface of sandy soil (South Africa, semi-desert); 30.1865 S, 17.5433 E
|
MZ546605
|
242
|
231/244 (95%)
|
HQ246450, and 4 other
|
BIOTA 153
|
Tetradesmus deserticola
|
Biological soil crust on the surface of sandy soil (South Africa, semi-desert); -30.3856 N 18.2757 E
|
ON677848
|
240
|
234/240 (98%)
|
AY510471
|
SAG 2608
|
Tetradesmus obliquus
|
Biofilm on soil surface next to a freshwater pond (Germany, Uslar); 51.647250 N, 9.761198 E
|
MZ546606
|
239
|
240/240 (100%)
|
MK975482, and 52 other
|
SAG 2607
|
Tetradesmus obliquus
|
Biofilm on soil surface next to a freshwater pond (Germany, Uslar); 51.647250 N, 9.761198 E
|
MZ546607
|
239
|
240/240 (100%)
|
MK975482, and 52 other
|
Growth under elevated CO2 atmospheres
All the 12 new green algal strains from terrestrial habitats exhibited robust growth under the atmospheres of elevated CO2. T. bajacalifornicus BIOTA 136 showed even enhanced growth at all tested levels of elevated CO2 atmospheres (growth pattern 7, Fig. 1). D. armatus SAG 2635 and D. multivariabilis SAG 2628 were tolerant to the 5 and 15 % CO2 atmospheres and exhibited enhanced growth under the 25 % CO2 atmosphere (growth pattern 6, Fig. 1). The new strains of T. obliquus, SAG 2607 and SAG 2608, had enhanced growth under 5 % CO2 but tolerated the higher CO2 levels (growth pattern 6, Fig. 1). In contrast, the growth of other species of Tetradesmus, i.e., T. arenicola and T. deserticola, was suppressed under 5 % and 15 % CO2 atmospheres, although it did not cease under 25 % CO2. Growth of the new C. vulgaris strains SAG 2606 and SAG 2629 showed nor or just few adverse effects under the tested elevated CO2 atmospheres (growth pattern 5, Fig. 1). Similar growth patterns, i.e., suppressed growth under the 15 % CO2 atmosphere, were found for the new strains of Chlamydomonas sp. SAG 2630 and P. aurantiaca SAG 2631 (growth patterns 4 and 2, Fig. 1).
Most other green algal strains already available from culture collections were also robust towards atmospheres of elevated CO2 conditions. Even at 25 % CO2, increased growth was observed for Desmodesmus komarekii strain CCAP 258/232 (growth pattern 6, Fig. 1). Most frequent were the growth patterns with no adverse effects at all three elevated CO2 levels, found in strains from all three tested classes of the Chlorophyta (growth patterns 5 and 4; Fig. 1). Among the already available green algal strains from the SAG culture collection, a number of those from the Chlorophyceae and Trebouxiophyceae tolerated just the 5% CO2 atmosphere while reacting with suppressed growth under higher CO2 levels, e.g., the tested strains of Ettlia carotinosa and Neocystis brevis (growth pattern 3, Fig. 1). Out of the Chlorophyceae, only Chlorococcum novae-angeliae strain SAG 5.85 reacted with suppressed growth under all atmospheres of elevated CO2 (growth patterns 2, Fig. 1). Among the tested strains of Trebouxiophyceae, only the growth of Chloroidium angusto-ellipsoideum, Coccomyxa avernensis, Lobosphaera incisa, Stichococcus ampulliformis, and two strains of S. bacillaris, was suppressed under elevated CO2 atmospheres (growth pattern 2, Fig. 1).
For all tested Cyanobacteria strains, CO2 atmospheres higher than 5% resulted in suppressed growth (growth patterns 3 and 2, Fig. 1). Two cyanobacteria strains did not grow, even under the 5 % CO2 atmosphere (growth pattern 1, Fig. 1). The tested strains representing unicellular red algae (Rhodophyta) had growth patterns under elevated CO2 atmospheres like those of the Cyanobacteria. In about half of the tested red algal strains, 5% was the only level of elevated CO2 tolerated, while all higher CO2 levels led to suppressed growth (growth patterns 3 and 2, Fig. 1). However, just for one single strain, Porphyridium purpureum SAG 1380-1a, the 25 % CO2 atmosphere did not affect its growth.
Among strains of stramenopile algae, class Eustigmatophyceae, the strains from terrestrial habitats of the genus Vischeria tolerated 5 % CO2, with V. polyphem strain SAG 38.84 even 15 % CO2 in the atmosphere (growth patterns 3 and 5, Fig. 1). However, the growth of the tested strains from marine phytoplankton, the genera Microchloropsis and Nannochloropis, was suppressed, or it even ceased at all elevated CO2 levels (growth patterns 2 and 1, Fig. 1). It confirms earlier studies on the CO2 utilization of N. oculata in response to CO2 aeration [23]. From the tested stramenopile algal strains of the class Xanthophyceae, only a single strain of a typical terrestrial (soil) alga, Heterococcus viridis SAG 2422, was left unaffected in growth until 15% CO2 in the atmosphere. However, Ophiocytium parvulum strain SAG 37.84 from an aquatic environment tolerated only the 5% CO2 atmosphere. The other tested Xanthophyceae strain reacted with suppressed or even ceased growth to elevated CO2 levels in the atmosphere (growth patterns 5, 3, 2, and 1, Fig. 1). Among the three tested strains of the diatom Phaeodactylum tricornutum, a diatom isolated from marine or brackish environments, the growth of two strains was suppressed under all tested CO2 levels (growth patterns 4 and 2, Fig. 1). Only one strain, SAG 1090-6, did not exhibit adverse effects on the growth under the 5 and 25 % atmospheres.
Productivity of selected green algal isolates of terrestrial habitats in submersed culture
We selected four of the new green algal isolates as examples to further test terrestrial microalgae (including one from a temporary freshwater rivulet) for their productivity of valuable compounds. To resemble processes in photobioreactors, we performed the tests in submersed culture, i.e., liquid culture medium that was aerated with CO2 at either ambient or 15% concentration. One selected strain was Tetradesmus bajacalifornicus SAG BIOTA 136, the only strain exhibiting enhanced growth under all elevated CO2 atmospheres (growth pattern 7, Fig. 1). Two more strains were T. obliquus SAG 2607 and SAG 2608, which differed in their tolerance towards 15 % CO2, i.e., with growth unaffected (SAG 2607) or slightly enhanced (SAG 2608) (growth pattern 6, Fig. 1). Finally, Chlorella vulgaris strain SAG 2606 represented those strains tolerating all three tested CO2 levels without change in growth (growth pattern 5, Fig. 1), the most common growth pattern among the tested green algal strains. We analyzed the effects along with CO2 levels from ambient to 15% in the aeration on the productivity of biomass and chlorophyll (Fig. 2), followed by the impact of aeration of 15% CO2 compared to that with ambient CO2 on the contents of carotenoid (Fig. 3) and total fatty acid contents (Fig. 4, top). Finally, we analyzed the same CO2 effects on the ten selected single fatty acids, which included 8 polyunsaturated fatty acids
(PUFAs; Fig. 4), measured as fatty acid methyl esters (FAMEs). A prerequisite for the tests in submerged culture was to ensure that the found effects would concern just those caused by the enhanced (15%) CO2 concentration. There is the possibility that changes in the pH value generated by the CO2 aeration may interfere as a selection criterion. However, only a relatively small alkalization was observed due to the CO2 aeration from ambient to 15% CO2 (Additional file 2: Fig. S2). The consumption of CO2 and nitrate largely overcompensated the potential acidification. The metabolism of both leads to alkalization of the medium, based on an H +- cotransport (in the case of nitrate) or Na +- cotransport (partly for CO2) via the plasma membrane. Therefore, the pH remains in the buffer area of the carbonate buffer system (pH 6.5) [22]. This also applies to cultivation on agar plates, the culture medium (20 mL) of which has the same buffer capacity as the liquid culture.
Increasing the CO2 supply stimulated biomass production in a liquid medium dependent on the CO2 concentration in all four strains (Fig. 2). A continuous increase in biomass with increased CO2 supply was observed for C. vulgaris and the two T. obliquus strains. In contrast, T. bajacalifornicus achieved its highest biomass productivity at 2% CO2 and there was no further increase with higher CO2 concentrations (Fig. 2). The chlorophyll content in the three Tetradesmus strains (Fig. 2) went up sharply at 1% CO2 to a level where almost no alterations occurred with further increasing CO2 concentrations. However, in T. obliquus SAG 2608, chlorophyll content decreased at 10 and 15% CO2, almost to the level under ambient CO2 concentration (Fig. 2). In C. vulgaris SAG 2606, there was a similar sharp increase to a level at which further supply of CO2 had hardly any effect.
Carotenoid contents under ambient CO2 (condition AC) were almost double as high or higher in the three Tetradesmus strains than in C. vulgaris (Fig. 3). Aeration with 15% CO2 (condition CC) nearly doubled the total carotenoid content in all four strains compared to ambient CO2 in air (condition AC). Carotenoid production may be a significant sink for excess carbon under elevated CO2 supply in these strains. Concerning total carotenoid content, C. vulgaris was less productive than the other three strains (Fig. 3).
Total fatty acid content, i.e., the sum of the ten measured fatty acids (Additional file 3: Table S1) under ambient CO2 (condition AC), was highest in T. bajacalifornicus compared to the other three tested strains (Fig. 4, top). Total fatty acid production increased with 15% CO2 (condition CC) in all four strains. The effect was most pronounced in C. vulgaris, where the entire fatty acid content almost doubled, whereas there was only a slight increase (about 15-30%) in the three Tetradesmus strains (Fig. 4, top).
We further investigated the content of 10 fatty acids, measured as FAMEs, in all four strains under ambient and elevated CO2 (conditions AC and CC; Fig. 4, Additional file 3: Table S1). C. vulgaris exhibited the highest 18:3α content. Also, C. vulgaris was most rich in the 16:3 fatty acid, followed by T. bajacalifornicus under both conditions, AC and CC. C. vulgaris differed from the other three strains that no 18:4 was found (Fig. 4). In all strains, an increase in 18:1 9z due to 15% CO2 aeration was observed. In general, the effect of elevated CO2 aeration on the measured fatty acids was most pronounced in C. vulgaris SAG 2606. There, the elevated CO2 gassing resulted in an increased content of all except the 16:4 measured fatty acids (Fig. 4). In contrast, in the three Tetradesmus strains, an increment due to elevated CO2 was found in only some fatty acids. In the three Tetradesmus strains there was little or no elevated CO2 (condition CC) effect for 16:0 and 18:2 LA with elevated CO2 (condition CC). In T. bajacalifornicus BIOTA 136 the contents of 16:3. 16:4, 18:3𝛼, and 18:4 fatty acids were increased while in the T. obliquus strains only those of the 18:0, and 18:1(n-9) (oleic acid) fatty acids were increased (Fig. 4). The positive effect for 18:4 clearly seen in T. bajacalifornicus was rather little in the two T. obliquus strains (Fig. 4).
Effects of macronutrient limitation on the productivity of the four selected terrestrial strains
Macroelement limitations for nitrogen and phosphorus were applied separately to test whether these manipulations could further increase or decrease the effects of elevated CO2 supply on the content of carotenoids and fatty acids. Under nitrogen limitation and ambient CO2 (condition A-N), the carotenoid content increased in all four strains compared to that obtained in the complete medium (condition AC) (Fig. 3). Aeration with 15% CO2 under nitrogen limitation (condition C-N) increased the carotenoid content in all four strains. However, phosphate starvation under ambient CO2 (condition A-P) did not affect total carotenoid contents in all four strains compared to complete medium (condition AC), i.e., they remained at about the same level. Elevated CO2, in addition to phosphate starvation (condition C-P), doubled the total carotenoid content compared to ambient CO2 in C. vulgaris and T. obliquus. However, it only slightly increased in T. bajacalifornicus (Fig. 3). Generally, the values obtained from phosphate starvation combined with elevated CO2 (condition C-P) were lower than those obtained under nitrogen limitation (condition C-N; Fig. 3).
Total fatty acid content was strongly stimulated in all four strains by N-limitation alone (condition A-N; Fig. 3) compared to their growth in complete medium (condition AC). However, the combined treatment of elevated CO2 and N-limitation (condition C-N) further increased total fatty acid content only in C. vulgaris (Fig. 3; Additional file 3: Table S1). From the detailed analysis, it was apparent that N-limitation alone, condition A-N, had a robust, increasing effect on 18:1 9z compared to the complete medium (condition AC) in all four strains (Fig. 4; Additional file 3: Table S1). However, the combined treatment, condition C-N, reduced the amount of 18:1(n-9) in the three Tetradesmus strains compared to N-limitation alone (condition A-N), but not in C. vulgaris where the combination still led to increased 18:1 9z content. Also, the amount of 18:0 decreased under the condition C-N in all four strains compared to that at ambient CO2 (condition A-N) (Fig. 4; Additional file 3: Table S1). There was an increase in the contents of 16:3 and 18:3α in all strains under the combined treatment (condition C-N). It was pronounced in C. vulgaris. The content of 18:4, the fatty acid was not found in C. vulgaris, and it was consistently higher under the combined treatment (condition C-N) in the two T. obliquus strains. The 18:4 fatty acid content was significantly increased in T. bajacalifornicus under the combined treatment (condition C-N). While N-limitation alone (condition A-N) raised the 18:0 fatty acid concentration considerably, the additional supply of CO2 (condition C-N) reduced its content (Fig. 4; Additional file 3: Table S1). The content of the 16:4 fatty acid was low in C. vulgaris under condition AC and not detectable under N-limitation, condition A-N (Fig. 4; Additional file 3: Table S1). In contrast, the Tetradesmus strains exhibited higher contents of the 16:4 fatty acids under the AC condition. Among those, nitrogen limitation reduced the 16:4 fatty acid content only in T. bajacalifornicus (Fig. 4; Additional file 3: Table S1).
P-limitation under ambient CO2 (condition A-P) only slightly increased total fatty acids compared to the complete medium, i.e., condition AC. (Figs 3, 4; Additional file 3: Table S1). In combination with elevated CO2 (condition C-P), fatty acid production was further expanded in C. vulgaris and T. obliquus but reduced in T. bajacalifornicus (Fig. 4). The absolute contents of all fatty acids under P-limitation corresponded quite well to their respective contents with complete media (Fig. 4). The detailed analysis showed a slight increase of content in all fatty acids (conditions A-P, C-P), except 18:1(n-9) and 18:0 in the T. obliquus and C. vulgaris strains (Fig. 4). At the same time, it decreased in T. bajacalifornicus (Fig. 4; Additional file 3: Table S1). We conclude that P-limitation had only negligible effects on the fatty acid contents. In all strains, the N-limitation (condition A-N) had the most significant impact on the contents of all ten tested fatty acids (Fig. 4). Especially, the 18:1 9z was increased under ambient CO2 (condition A-N) (Fig. 4; Additional file 3: Table S1).