2.1. Habitat of microalgae
The microalgal sample isolated from the stagnant seawater of Kanyakumari district, Tamil Nadu (Latitude: 8° 7'28.10"N; Longitude: 77°29'16.89"E) was identified as Picochlorum sp. [Strain name: Picochlorum sp. NITT 04], which was characterized by tiny, unicellular, non-flagellated microalgae about ~ 2 µm in diameter (Table 1). The microalgal sample collected from the saltpan of Kanyakumari, Tamil Nadu (Latitude: 8° 6'38.91"N; Longitude: 77°29'13.43"E) was morphologically identified as Chlorella sp. [Strain name: Chlorella sp. NITT 02] characterized by unicellular, non-motile, round shaped microalgae. The other sample isolated from stagnant seawater in Muttukadu, Chennai (Latitude: 12° 48’ 45. 76” N; Longitude: 80° 14’ 37. 67” E) was morphologically identified as Chlorella sp. [Strain name: Chlorella sp. NITT 05]. Identified microalgal strains were initially grown in 50 mL conical flasks containing ASN-III medium and the temperature was maintained at 25ºC ± 2ºC with a light intensity of 1500 lux for 16:8 h light: dark photoperiod. All the primary stock cultures of isolated marine microalgae are maintained under the above-mentioned culture conditions and scaled up further to generate high voluminous biomass for subsequent studies.
Table 1
Marine microalgal isolates and their geographical sites in South East Coast, India
Isolate No.
|
Strain name
|
Habitat
|
Collection site
|
GPS
|
Isolate − 2
|
Chlorella sp.
NITT 02
|
Saltpan
|
Kanyakumari, Tamil Nadu
|
8° 6'38.91"N
77°29'13.43"E
|
Isolate − 4
|
Picochlorum
NITT 04
|
Stagnant seawater
|
Kanyakumari
Tamil Nadu
|
8° 7'28.10"N
77°29'16.89"E
|
Isolate − 5
|
Chlorella sp.
NITT 05
|
Stagnant seawater
|
Muttukaddu, Chennai
|
12° 48’ 45. 76” N
80° 14’ 37. 67” E
|
2.2. Asynchronous growth metrics of microalgal strains
Fixed inoculum density of Picochlorum sp. NITT 04, Chlorella sp. NITT 02, and Chlorella sp. NITT 05 was analyzed for time course asynchronous growth kinetics from day 0 to 27 using triplicate experiments (Fig. 1). Of the strains tested, Picochlorum sp. NITT 04 was found to show higher cell density at about 2.23 OD on 27th day (Fig. 1c). The growth of Picochlorum sp. NITT 04 was gradually increased from day 0 till day 27; however after 27th day of its growth, no remarkable trend in growth rise was noticed. Similar to the results reported from this study, the OD value of Picochlorum oklahomensis after 25th day was around 2 and subsequently decreased over time 11. In the case of Chlorella strains, the initial OD of the Chlorella sp. NITT 02 and NITT 05 culture was 0.61 on day 0 and the OD got increased during the growth and the high culture density (maximal OD) was seen on cultivation day 27 which is 1.45 and 1.62 OD, respectively (Fig. 1a, 1b). The observation in Chlorella sp. NITT 02 & NITT 05, agrees to some extent with the study conducted by Chioccioli et al., (2014) demonstrating that Chlorella vulgaris reached stationary phase at around OD750 value of 1.3 when grown in TAP medium. Also, various recent reports in the growth pattern of different Chlorella sp. were in accordance with the results obtained from this study 13–16. The cell density obtained in this study was higher since the culture was grown in ambient condition without CO2 purging. It could rise further by cultivating it in high strength medium purged with CO2.
2.3. Lipid extraction by Switchable solvent system
In the present study, growth metrics of Picochlorum sp. NITT 04, Chlorella sp. NITT 02, and Chlorella sp. NITT 05 disclosed that higher biomass was produced on 27th of cultivation, but, no information on lipid content and yield has been retrived, which is considered to be a important parameter for biodiesel production. Therefore, it is imperative to study lipid content of the strain to evaluate its feasibility for biodiesel and the lipid extraction was done using the biomass harvested on 27th day. Switchability nature of tertiary amine during lipid extraction from marine microalgae is illustrated in Fig. 3. The amount of lipid extracted by the solvent (fresh tertiary amine) indicated as “fresh” and the amount of lipid extracted by the solvent after first use, indicated as “recovered” in Fig. 4a were calculated gravimetrically. It is apparent that DMBA solvent was able to extract more lipids than DMCHA and DIPEA solvents. Also, Chlorella sp. NITT05 yielded significantly more lipids followed by the other two species. It is to be noted that, higher lipid at about 42% was extracted by both fresh and recovered solvent of DMBA from Chlorella sp. NITT05 whereas it was 23% using DMCHA. The other strain Chlorella sp. NITT02 yielded 27% with the use of fresh and recovered DMBA solvent. In the case of Picochlorum strain, higher lipid content of 33% was obtained from DMBA and 23% was obtained from DMCHA. On a similar note, lipids extracted from freeze dried Botryococcus braunii by DMCHA accounted to 22% of crude lipid yield 17. Another study by Samorì et al., (2013) reported that the total lipid content of D.communis, N.gaditana, and T.suecica by DMCHA extraction was 29.2%, 57.9%, and 31.9% respectively. Among the tertiary amines used, the DIPEA was found to be inefficient in extracting lipids from all the strains and lipids extracted by DIPEA solvent was very less compared to other two solvents and restricted to switchability by forming salts after CO2 purging. This agrees with the existing literatures, where the formation of solid carbamate salts hinders the reversion process by taking great time and requiring high temperature for conversion. Hence, the choice is over liquid amines resulting in liquid carbamate salts allowing the ease of switchable nature from liquid carbamate salts which forms the basis of SPS 19,20. The lipids extracted by DIPEA were 10, 12.12 and 13.3% for Picochlorum sp. NITT 04, Chlorella sp. NITT 02, and Chlorella sp. NITT 05, respectively. Hence from the observations, lipids extracted from Chlorella sp. NITT05 and Picochlorum sp. NITT 04 by DMBA is more sustainable in terms of both lipid and solvent recovery. In addition to the tertiary amines, secondary amines such as 2-Methylaminoethanol, 2-Ethylaminoethanol, Diisopropylamine and Diethylamine were also checked for its ability to extract lipids. But, all the secondary amines listed above formed a monophasic system (amine and microalgal sample as a single phase) instead of biphasic system (amine and microalgal sample as different phase) as observed in tertiary amines, thereby leading to difficulty in separating lipids from amines and also in switching from hydrophobic to hydrophilic and vice-versa. Hence, lipid extraction from wet microalgal suspension was achieved through tertiary amine as switchable solvent. For switching from hydrophobic state to hydrophilic state, all the three tertiary amines were purged with CO2. After CO2 purging, the switching of hydrophilic tertiary amine to hydrophobic amine differed for each of the tertiary amines used. For DMBA, hydrophilic to hydrophobic state was achieved through N2 purging. For DMCHA, it was achieved through constant stirring and heating at 80ºC. For DIPEA, switching was difficult as it led to formation of salts after exposure to CO2. During lipid extraction, amines establish interaction with the available lipid molecules preferably after disruption of cell wall. Neutral lipids on the other hand form globules in the cytoplasm as they are of long hydrocarbon chains and when amine enters the cell, it forms vanderwaals interaction with neutral lipids and transfers from the cell through concentration gradient 20. Solvents generally work by the principle of ‘like dissolves like’. Amines with adequate ‘alkyl’ groups behave as non-polar solvents. In switchable solvent system, amines react with CO2 to form corresponding carbamic acids as an intermediate and further gets converted to corresponding carbamate salts. At this point of time, amines behave as polar solvents; upon triggering by bubbling argon or nitrogen gas, it reverts to its original form 21. The reaction between amine and CO2 is understood by zwitterion mechanism. In this mechanism, amines, which are basic in nature reacts with acidic CO2 gas and forms an intermediate zwitterion consisting of pool of both the charges. In a consecutive step, the protons from the zwitter ion intermediate transfers to a second molecule known as a base and becomes protonated, thus leaving carbamate ions, where base can be amine, hydroxyl ions, water or alcohol. Another well-known mechanism is intermolecular reaction mechanism which takes place in a single step. Amines, base, and CO2 react together in a single step without any intermediate formation, resulting in carbamate ions and protonated base 22.
(1)
In case of tertiary amines, bicarbonate salts which are soluble in water are formed when exposed to CO2. The switching of tertiary amine is illustrated by the following reaction ,Eq. (1) 9. In this regard, it is noteworthy to mention that, DMBA is determined to be an efficient and competent tertiary amine for microalgal lipid extraction in terms of extraction performance, and easy switching (energy efficient processes like heating and stirring not required).
2.3.1. Solvent recovery and reuse
The tertiary amines DMBA, and DMCHA recovered after first extraction of lipid was subsequently used for extraction of lipids from the wet microalgae for the second cycle. The solvent recovery is presented in Fig. 4b and it is inferred that the overall solvent recovery for both DMBA and DMCHA is less than 30% and varied slightly between the microalgal species. Among the two tertiary amines, DMBA is considered as a potential solvent in terms of both lipid extraction and solvent recovery followed by DMCHA. In concern with DIPEA, it was difficult to recover the amines after first extraction and therefore, the recovered solvents of DMBA (23–28%) and DMCHA (25–30%) were used for second cycle of extraction. Though the solvent recovered was in minimal quantity, it retains its lipid extracting efficacy as the lipid content extracted from fresh and recovered solvent was similar as evident from Fig. 4a.
2.4. TLC for qualitative estimation
Qualitative determination of lipid types present in switchable solvent extracted total lipids was carried out. To ensure the presence of neutral lipids in DMBA, DMCHA, and DIPEA extracted total lipids, TLC was carried out. Further, only one strain i.e., Picochlorum sp. NITT 04 was chosen for TLC experimentation as TLC refers to be qualitative separation and therefore, lipids of all strains were subjected for GC analysis to quantify the fatty acid composition. As shown in Fig. 5, the presence of neutral lipid (Picochlorum sp.) in TLC is encircled in all solvent types used. Based on i) the presence of neutral lipid ii) solvent recovery and iii) lipid content and (iv) switchability phenomenon, DMBA is considered as ideal solvent for lipid extraction.
2.5. Fatty acid compositional analysis of microalgae
The predominant fatty acids present in Chlorella sp. NITT02, Chlorella sp. NITT05, and Picochlorum sp. NITT04 was presented in Table 2. Among the fatty acids, the 16 carbon long chain palmitic acid is the found to be predominant at about 24.77, 29.4, and 28.32% in Chlorella sp. NITT 02, Chlorella sp. NITT 05, and Picochlorum sp. NITT 04, respectively. Palmitic acid is one of the major saturated fatty acids which are commonly found in many microalgal species. The difference among the fatty acid dominance for all the three strains was clearly visualized based on color-coding in Heat map (Fig. 6). The second most predominant fatty acid present in tested strain was oleic acid (C18:1), which accounts 14.4, 17.7, and 18.5%, respectively for Chlorella sp. NITT 02, Chlorella sp. NITT 05, and Picochlorum sp. NITT 04 among the total fatty acid composition. In fact, many studies report the same type of fatty acids to be the major component in the total FAME content 15,23−25. As graphically represented in heat map, of the strains analyzed for FAME, Chlorella sp. NITT 02 possesses high levels of polyunsaturated fatty acids (PUFA) such as linoleic acid, linolenic acid, and Cis-13,16- docosodienoic acid, which contributes 27% in total fatty acid composition of the strain. In contrast, Chlorella sp. NITT 05 accumulates saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) maximally. It is noteworthy to say that the presence of higher amount of SFAs than PUFAs makes the biodiesel less incline towards rancidification. In the case of Picochlorum sp. NITT 04, it is interesting to note that, monounsaturated fatty acids were present in higher concentration along with lower levels of PUFA. These results are similar to the studies conducted by El-kassas, (2014) and Yang et al., (2015) where high amounts of MUFAs are observed than the polyunsaturated fatty acids for marine Picochlorum sp. The single double bond containing fatty acid types namely oleic acid and palmitoleic acid accounts at about 18.5 and 8.5%, respectively in Picochlorum sp. of this study and further, linolenic acid content was estimated to be less than 12%. This fatty acid content is almost similar to the fatty acid content of Picochlorum oklahomensis where 13.85%, 8.2%, and 13.52% of oleic acid, palmitoleic acid, and linolenic acid resepectively were observed 11. From Table 2, it is understood that maximal concentration of SFAs and MUFAs and minimal concentration of PUFAs were observed in both Chlorella sp. NITT 05 and Picochlorum sp. NITT 04. Likewise, 32.8% of SFAs and 62.3% of total monosaturated and disaturated fatty acids were observed in Picochlorum sp. under ambient conditions 27. Fatty acid composition decides the fuel properties of biodiesel. The fuel properties to be assessed for using biodiesel as a fuel are oxidative stability, cetane number, viscosity, cold filer plugging point or low temperature property, flash point, pour point, ash content, total glycerol content, calorific value etc 28. Higher concentration of SFAs is beneficial for biodiesel as they determine the oxidative stability of the fuel and higher SFA content is linearly proportional to oxidatively stable biodiesel. Also, higher cetane number (CN) is obtained by the presence of high level of SFAs where CN determines the ignition potential i.e., higher CN, less time for ignition and vice versa. At the same time, presence of linolenic acid greater than 12% causes the CN to be very low, thus making the quality of biodiesel to be at low standard 29. The FAME profile of the tested microalgal species in this study satisfies the above-mentioned properties of biodiesel standard. On the other hand, high level of SFAs increases the viscosity as well as raise Cold filter plugging point (CFPP) thus, claiming the biodiesel to be unsuitable to operate under low temperature 30. Overall, there should be optimal balance of saturated and unsaturated fatty acids to qualify the properties listed for biodiesel standard. Autooxidation of the fuel relies upon the double bond present in the fatty acids. Increase in the double bond numbers (PUFA) in fatty acids makes the biodiesel susceptible to autoxidation. Compared to allylic positions, bis-allylic positions in the fatty acid chain are susceptible to autooxidation. And it is widely known that linoleic acid contains one bis-allylic position and linolenic acid contains two bis-allylic position which is why their concentration in the total FAME content is supposed to be less 31.
Table 2
Fatty acid methyl ester composition of Chlorella sp. NITT02 Chlorella sp. NITT05, Picochlorum sp. NITT04
Fatty acids
|
Carbon number and double bond of fatty acids
|
Percentage of fatty acids (%) in total FAME pool
|
Chlorella sp.
NITT02
|
Chlorella sp.
NITT05
|
Picochlorum sp. NITT04
|
Lauric acid
|
C12:0
|
5.98
|
6.470
|
3.230
|
Tridecanoic acid
|
C13:0
|
3.94
|
4.030
|
3.120
|
Myristic acid
|
C14:0
|
Lower conc.
|
Lower conc.
|
3.280
|
Palmitic acid
|
C16:0
|
24.77
|
29.430
|
28.320
|
Palmitoleic acid
|
C16:1
|
8.87
|
10.670
|
8.550
|
Oleic acid
|
C18:1
|
14.43
|
17.710
|
18.500
|
Linoleic acid
|
C18:2
|
10.65
|
4.090
|
3.920
|
Linolenic acid
|
C18:3
|
12.05
|
11.450
|
9.130
|
Cis-11,14-eicosodienoic acid
|
C20:2
|
3.28
|
4.150
|
3.570
|
Cis-11,14,17- eicosotrienoic acid
|
C20:3
|
3.55
|
3.180
|
4.180
|
Cis-13,16- docosodienoic acid
|
C22:2
|
4.34
|
3.080
|
3.440
|
Tricosonoic acid
|
C23:0
|
3.76
|
3.110
|
3.030
|
Fatty acids detected in trace concentration
|
-
|
4.38
|
2.63
|
7.710
|