High-Efficiency Ukrainian Strains of Microalgae for Biodiesel Fuel Production (Overview)

3.1. Screening of IBASU-A Strains Collection for Promising Candidates for Algal-Based Biodiesel Production and Comparison with Strains from other Collections

At the initial stage of our studies, we screened the strains present in IBASU-A collection to identify promising candidates for biodiesel production. According to original and published data, the main criteria for this selection were: the active growth of algae, their tolerance to stress factors and resistance to biological contamination and accumulation of a rather significant amount of lipids. To select the fast-growing strains, the express-method, including algal cultivation under uniform culture growing conditions with visual determination, was initially used [29]. Then a study of the growth characteristics of selected strains was carried out under intensive conditions during 10 days at temperature 26-32°C, constant light intensity of 100 µmol m^-2 s^-1 and constant aeration. The initial cell density in the flask was 5·10⁶ cells mL^-1. The daily increase of algal biomass was estimated by microscopic cell counting or determining dry cell weight (DW) gravimetrically [30]. The growth characteristics (specific growth rate and biomass productivity) were calculated according to the formulas [31] on the basis of the obtained data. They were compared with those of known high biomass producers Tetradesmus dimorphus (Turpin) M.J. Wynne IBASU-A 254 (=Scenedesmus acutus Meyen CALU 24; = Acutodesmus dimorphus (Turpin) P. Tsarenko), and Ch. vulgaris IBASU-A 189 (= CALU 157) obtained from CALU (St-Petersburg, Russia).

As a result of this screening, a new collection of strains as potentially high biomass producers was established. Overall, it included 33 strains of 18 species from Botryococcus Kütz. (1), Chlorella Beijr. (5), Chloroidium Nadson (2), Desmodesmus (Chodat) S.S. An, T. Friedl et E. Hegew. (9), Euglena Ehrenb. (2), Monoraphidium Komárk.–Legner. (2), Parachlorella Krienitz, E. Hegew., Hepperle, Huss, Rohr et Wolf (6) and Tetradesmus G.M. Sm. (=Acutodesmus (E. Hegew.) P. Tsarenko) (6 strains) genera. This new collection included strains obtained from other collections (15 strains from CALU, UTEX, SAG, CCAP and CCALA) or isolated from territories of other countries (7) and Ukraine (10).

The optimal media and growing conditions for these strains were determined before carrying out further experiments.

The comparative studies on the growth characteristics of selected strains showed that the majority of them demonstrated a rather active growth [32]. The maximal cell concentration (B) was shown by strains of Ch.vulgaris IBASU-A 189, 190, 192, 326, 452, Chloroidium saccharophilum IBASU-A 186, 187 and Parachlorella kessleri IBASU-A 197-201, 444 and ranged from 38·10⁶ to 250·10⁶ cells mL^-1, whereas their specific growth rate (µ) was in the range of 0.55-1.4 day^-1 and biomass productivity (P) in the range of 9.5-72.5·10⁶ cells mL^-1 day^-1. A little less was the maximal cell concentration obtained for species of Desmodesmus (IBASU-A 270, 310, 341, 371, 384, 398, 401, 402, 407), and Tetradesmus (T. dimorphus IBASU-A 251, 252, 344, T. obliquus (Turpin) M.J. Wynne IBASU-A 292, 473) varying in the range of 26-84.5·10⁶ cells mL^-1, with the specific growth rate ranging from 0.35 to 1.2 day^-1 and the biomass productivity from 6.4 to 29·10⁶ cells mL^-1 day^-1. There was no obvious adaptation phase in cultures of all strains under study. The stationary phase was observed at the fifth or sixth day from the beginning of experiments for the majority of strains. The presence of the satellite bacteria did not influence the growth characteristics of the algal strains under study. Under optimal conditions of batch cultures, the biomass productivities in the strains of Chlorella, Chloroidium and Parachlorella species varied in the range of 0.51-1.6 g DW L^-1 ·day^-1 and in those of Desmodesmus and Tetradesmus species in the range of 0.34-1.2 g DW L^-1·day^-1. The biomass production of Botryococcus braunii IBASU-A 504 reached 1.3 g DW L^-1·day^-1. Monoraphidium griffithii (Berk.) Komárk.-Legner. (IBASU-A 166, 364) and Euglena viridis (O.F. Müller) Ehrenb. (IBASU-A 496, 498) species had a rather high level of productivities, up to 0.29-0.38 g DW L^-1·day^-1.

Eight strains, including control Ch. vulgaris IBASU-A 189 and T. dimorphus IBASU-A 254 were identified as the highest productive ones since they showed the increase of biomass in the range of 0.58 ±0.03 -1.6 ±0.26 g DW L^-1·day^-1. They were Desmodesmus magnus (Meyen) P. Tsarenko IBASU-A 402, D. multivariabilis var. turskensis P. Tsarenko et E. Hegew., Ch. vulgaris IBASU-A 190 (=CALU 158), P. kessleri IBASU-A 197 (UTEX 397), P. kessleri IBASU-A 198, and T. dimorphus IBASU-A 251 [33].

According to the published data, a number of promising biomass and lipid producers have been found among the representatives of families Chlorellaceae (Auxenochlorella (I. Shihira et R.W. Krauss) T. Kalina et M. Punčochárová, Chlorella, Chloroidium, Parachlorella) and Scenedesmaceae (Desmodesmus, Scenedesmus, Tetradesmus (=Acutodesmus) [12, 13, 34-37].

Mahmoud et al. [35] reported the strains of Ch. vulgaris and “Scenedesmus quadricauda” isolated from different localities of Egypt with the biomass productivity of 1.23-1.09 g DW L^-1·day^-1 and lipid content of 37% and 34% of DW, respectively.

Aboun-Shanab et al. [36] reported the strain “Scenedesmus obliquus” YSR01 isolated in China from a fresh water body with a growth rate of 1.68 day^-1, biomass productivity of 1.57 g DW L^-1·day^-1 and total lipid content 58% of DW [37].

Liu et al. [12] studied 43 algal strains from the FACHB collection; China isolated and identified as members of Chlorellaceae and Scenedesmaceae families for biofuel production purposes. In the course of a wide-scale screening of Chinese algal strains, 10 promising candidates for biofuel applications were selected according to three important parameters: total lipid content, biomass productivity and lipid productivity. The biomass productivity of the tested strains ranged from 0.53 g DW L^-1·day^-1to 6.07 g DW L^-1·day^-1, with the highest biomass of 6.07 g DW L^-1·day^-1for S. bijugatus. The lipid content for all strains varied from 20% to 51% of DW. “Chlorella pyrenoidosa” Strain 4 was considered the best oil producers since it showed the best combination of biomass productivity and total lipid content. Under optimal conditions (BG-11 medium, bubbling air with 0.5% CO², light intensity 150 µmol m^-2 s^-1) algae accumulated 3.02 g DW L^-1·day^-1, with total lipid 51.28% of DW.

Although research on this field is mainly focused on testing the oil-producing strain capacity in outdoor cultivation under stress conditions such as changing light intensity, pH, temperature, salinity etc, we believe that two additional criteria should be considered: resistance to stresses and biological contaminations. Many researchers use the first one in preliminary experiments for the determination of optimal growth conditions for experiments; however, the second one is often left out of consideration. Regardless of high growth parameters and lipid content, the use of some strains is challenged because of their vulnerability to invasion by other algae, bacteria or fungi. The species of Chlamydomonas have been excluded from our list of promising strains due to their high sensitivities to contamination by the small-celled green Chlorella-like and green-blue algae, which were evident under cultivation. Some strains of Botryococcus also appeared to be highly sensitive to biological contaminations.

Our experiments on the oil accumulation included the determination of total lipid content and the fatty acid profile as two very important parameters for the selection of strains for biodiesel purposes. The strains Ch. vulgaris IBASU-A 189, 190, P. kessleri IBASU-A 444 and T. dimorphus IBASU-A 344 were cultivated under normal growing conditions without nitrogen deprivation [13, 14, 16, 38], phosphate limitation [39], high salinity [14, 16, 40], high light intensities [41] and iron content of the media [42] to improve the lipid content. Only for the T. dimorphus IBASU-A 251, an additional experiment was carried out to determine the impact of nitrogen deprivation on total lipid concentration and fatty acid profiles. Accordingly, T. dimorphus IBASU-A 251 was cultivated in two stages: the first two weeks on full media and the second two weeks on media without nitrogen.

For the determination of total lipids, samples (1 g of freeze-drying algal biomass) were extracted with isopropanol, then by a mixture of isopropanol: chloroform (1:1, v/v), followed by a mixture of chloroform: methanol (1:1, v/v) repeated twice. The weight of the crude lipid obtained was determined gravimetrically. The fatty acids profile of the extracted lipids was determined as methyl esters (FAMEs). The FAME composition was determined by the gas-liquid chromatography method using a Gas Chromatography GC-16A Shimadzu (Japan) with a flame ionization detector and the GS solution software. A capillary column THERMO TR-FAME (30 mm×0.25 mm ID×0.25 mm film) with a temperature gradient from 70 to 230°C was used for a split. Helium was used as carrier gas at a flow rate of 1 mL∙ min^-1. The temperature of the injector and detector was 280 and 260°C, respectively. The identification of the fatty acids was carried out by comparing the retention time of the extracted compounds with that of standards. The content of fatty acids was expressed as a percentage of total dry weight [43].

Overall, under standard growing conditions, the total lipid content was 16.9±0.7% and 17.5±1.2% of DW for Ch. vulgaris IBASU-A 189, 190, respectively; 10.53±0.5% of DW for P. kessleri IBASU-A 444 and 12.4±2.5% and 17.5±3.8% of DW for T. dimorphus IBASU-A 251 and 344, respectively (Fig. 2 a, b). The total lipid content of 19.8±2.4% of DW was obtained for algal biomass of T. dimorphus IBASU-A 251 grown under nitrogen-deprivation; thus lipid content increased up to 37.4% of DW under nitrogen deprivation with respect to standard growing conditions [39]. This observation is in line with the data from other researchers who investigated the effects of nitrogen deprivation on the lipid content of green microalgae from Chlorellaceae and Scenedesmaceae [13, 16].

The chemical characteristics of fatty acids, such as carbon chain length and unsaturation extent determine biodiesel properties. Therefore, it is necessary to identify fatty acid profiles of the most promising algal strains. Palmitic (C16:0), oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) are recognized as the most common fatty acids in microalgal lipids [22]. Our results showed that all these saturated, monounsaturated and polyunsaturated fatty acids were produced by the strains under study (Fig. 3). The most abundant fatty acids found in Ch. vulgaris IBASU-A 189, 190 were C16:0 – 21.28% and 18.94%, C18:2 – 20.92% and 26.82% and C18:3 – 26.48% and 26.48%, respectively. The same fatty acids were found in P. kessleri IBASU-A 444 – C16:0 – 16.32%, C18:2 – 26.38% and C18:3 – 22.80%. Ch. vulgaris IBASU-A 189, 190 and P. kessleri IBASU-A 444 also produced palmitoleic (C16:1) and oleic (C18:1) acids but in small amounts. On the contrary, in T. dimorphus IBASU-A 251, 344 most abundant fatty acids were C18:1 – 21.80% and 20.96%, less abundant – 11.41% and 12.41%, respectively, and minor acids C18:0, C18:2 and C18:3 were also found. Under nitrogen-deprivation in the culture of T. dimorphus IBASU-A 251, the quantity of dominant acids C16:0 increased twice, C18:1 up to 34%; minor acids C18:2 – one and a half and C20:1 – in three times [43].

Thus, the screening of promising candidates for the algal-based production conducted on IBASU-A collection allowed to reveal five high-efficiency strains belonging to genera Chlorella, Parachlorella and Tetradesmus, which were capable of accumulating biomass more than 1.2 g DW L^-1·day^-1. While the total lipid contents for them varied from 10.53% to 19.8% of DW, the composition of fatty acids in the selected strains was mainly C16:0, C18:2 and C18:3. They were considered to have significant potential for biofuel applications, but further investigations on improvements in culture conditions to elevate their lipid productivities have to be conducted.

3.2. Screening of Wild Microalgae of Family Selenastraceae Adapted to Regional Environmental Conditions

During the years 2013-2018 48 strains of Chlorellaceae, Scenedesmaceae and Selenastraceae families were isolated to enrich the IBASU-A collection with Ukrainian strains from different climatic conditions. They were isolated mainly from inland shallow mesotrophic water bodies from the different physical and natural geographical regions (forest, steppe-forest and steppe zones). Among them eight strains of species were found and further investigated from Monoraphidium, Raphidocelis Hindák, Selenastrum Reinsch (Selenastraceae) genera that have currently attracted more attention of researchers worldwide [44-52].

The perspective of use and the need to study Selenastraceae-related strains have been most fully considered by Bogen et al. [44, 45]. A detailed investigation of some species from the genus Monoraphidium (M. arcuatum, M. contortum, M. dybowski, M. griffithii, M. neglectum, M. terrestre and M. tortile, all strains from SAG) was conducted in regard to biomass productivity, lipid amount and lipid profile as well as biomass degradability. The mean increase of biomass of strains was measured by the determination of dry biomass after the cultivation period, divided by the number of days of cultivation. It ranged from 0.033 g DW L^-1·day^-1 in M. tortile SAG 16.81 to 0.360 g DW L^-1·day^-1 in M. terrestre SAG 49.87. While total lipids content varied in the range of 15.9-31.5%. The strains of M. terrestre SAG 49.87 M. dybowski SAG 202-7e and M. contortum SAG 47.80 showed the highest biomass productivities; the highest total lipid content was found in M. tortile SAG 16.81.

Shrivastav et al. [51], studied a Monoraphidium sp. microalga isolated during the mass cultivation of Ettlia sp. for its biodiesel production properties. The biomass, lipid productivity and fatty acid profile of a new microalgal strain were examined under autotrophic cultivation conditions using various CO₂ concentrations and mineral and organic nitrogen sources. This new strain was found as a promising feedstock for biodiesel production due to its high lipid content (28.92% of DW) and increase of C18:1 amount under cultivation with urea as a source of nitrogen.

Patidar et al. [50], also considered Monoraphidium minutum (Nägeli) Komárk.-Legner. for carbon sequestration and lipid production in response to varying growth modes.

Indeed, a previous study reported the use of Monoraphidium sp. for biodiesel production. As a matter of fact, Yu et al. [14], identified Monoraphidium sp. FXY-10 strain, isolated from Lake Fuxian in China, as a potential candidate for biodiesel production purposes. This strain had high growth and total lipid content up to 56.8% of DW under photoautotrophic conditions. Moreover, the cultivation of FXY-10 under heterotrophic conditions led to increase lipid productivity from 6.88 mg L⁻¹ day⁻¹ to 148.74 mg L⁻¹ day⁻¹ and the production of saturated and monounsaturated fatty acids up to 77.5%. Another Monoraphidiumsp strain, QLY-10, was isolated from Qilu Lake in Yunnan Plateau (China) and used for developing a two-step strategy for algal biomass and lipid production [46].

During our investigations for algal strains adapted to climatic conditions, eight strains of Selenastraceae family were isolated by conventional methods: cultivation of algae on enriched selective media followed by single-cell isolation using either a micropipette or agar plate techniques [29]. For taxa identification, both morphological and molecular-biological analyses were used. The last consisted of RAPDs (Random Amplified Polymorphic DNA) and TBP-method based on the conservatism of the exon sequences of β-tubulin genes in all eukaryotic organisms [53, 54].

The comparative studies by Nygaard et al. conducted on the growth characteristics of Monoraphidium griffithii IBASU-364, 493, M. minutum IBASU-A 574, Monoraphidium sp. IBASU-A 377, Raphidocelis subcapitata (Korschikov) Nygaard et al. IBASU-A 358, 360, Selenastrum gracile Reinsch IBASU-A 317, 580 and Ch. vulgaris IBASU-A 189 as a control, revealed that most of them possessed active growth, high specific growth rate (µ), and biomass productivity (P) (Table 1 ). There was no obvious adaptation phase in cultures of all strains under study. The stationary phase was observed at the fifth or sixth day from the beginning of experiments for the majority of strains. In comparison with Ch. vulgaris 189, the most productive strain was M. minutum 574 with increasing biomass of 1.84 g DW L^-1·day^-1, followed by R. subspicata 358, 360 with 1.32-1.48 g DW L^-1·day^-1 and Monoraphidium sp. 377 with 0.84 g DW L^-1·day^-1. Generally, the newly isolated Ukrainian strains demonstrated rather high growth characteristics such as the specific growth rate (0.38-1.2 day^-1) and biomass productivity (0.31-1.84 g DW L^-1·day^-1) (Table 2, Fig. 2c). The total lipid contents of the three most productive strains M. minutum 574, Monoraphidium sp. 377 and R. subcapitata 358 were 33.65±0.87%, 29.43±1.07% and 23.14±1.25%, respectively. We note that the lipid content in the Monoraphidium spp. cultured under standard conditions was in the range of 19.9–43.5% [19].

A study of fatty acid composition of these strains showed a certain variability of dominant fatty acids in different strains (Fig. 3). The dominant fatty acids found in M. minutum 574 were C18:2 – 29.17% and C18:3 – 29.72%, in Monoraphidium sp. 377 – C16:0 – 28.32%, C18:1 – 38.86% and C18:2 – 14.63%, and in R. subcapitata 358 – C18:1 – 61.28% and C18:2 – 19.48%.

Our preliminary data about the fatty acid composition of Monoraphidium species are in accordance with those of other researchers. According to Li et al. [49], the fatty acid profile of Monoraphidium sp. QLY-1 (China) included dominant C16:0, C18:1, C18:2, and C18:3. The same dominant fatty acids were found in M. minutum isolated from freshwater lagoon from Hazira Surat (India) and their content varied under different experimental conditions [51]. The dominant fatty acids of Monoraphidium sp. FXY-10 (China) were C16:0, and C18:3 in autotrophic and C16:0 and C18:1 in heterotrophic cultures [14]. A cold-adapted strain of Monoraphidium sp. isolated from an Antarctic ice-covered lake produced a high amount of polyunsaturated fatty acids C16:4 and C18:4 under low temperature conditions [19].

As shown by Bogen et al. [44], all species of Monoraphidium (M. arcuatum, M. contortum, M. dybowski, M. griffithii, M. neglectum, M. terrestre and M. tortile) under study had the fatty acid profiles with high relative amounts of C16:0, ranging from 16.5% in M. dybowski SAG 202-7e to 27.3% in M. neglectum SAG 48.87 and high contents of C18:1 fatty acids of up to 57.6% in M. neglectum. It has been revealed that there is a link between the prevalence of C16:0 and C18:1 fatty acids and the maximum growth phase when algae are harvested. The increasing contents of these fatty acids were also observed during nitrogen starvation. Thus, it can be argued that specific nitrogen starvation conditions might enhance the levels of useful lipids.