The biomass productivity, lipid content, lipid productivity, N-removal rate (NRR), N-removal efficiency (NRE), P-removal rate (PRR), and P-removal efficiency (PRE) of five different green algal species concerning varying N:P (0.53 to 6.84) in BBM are found out. The experiments examined variations in N and P concerning N:P in BBM on biomass productivity, lipid content, lipid productivity, and N and P removal rate and efficiency concerning the five species of algae. The highest Biomass Productivity (BP), Lipid Yield (LY), and Lipid Productivity (LP) of the five species of algae concerning N or P or N and P together, and N:P are shown in Table 2. The details of the best NRE, NRR, PRE, and PRR concerning N or P or N and P together, and N:P in BBM by the five species are shown in Tables 3 and 4, respectively.
Table 2
The highest Biomass Productivity (BP), Lipid Yield (LY), and Lipid Productivity (LP) of the five species of algae
Algae
|
Exp
|
Treatment
|
N:P
|
Biomass productivity (BP)
|
Treatment
|
N:P
|
Lipid yield (LY)
|
Treatment
|
N:P
|
Lipid productivity (LP)
|
---|
Monoraphidium contortum
|
1
|
BBM &BBM2N
|
1.71to 3.42
|
77.69 ± 1.53to 80.72 ± 1.55
|
BBM
1/2N
|
0.85
|
44.38 ± 0.81
|
BBM
|
1.71
|
31.52 ± 1.06
|
2
|
BBM2P,BBM3P&BBM4P
|
0.98 to 0.53
|
102.22 ± 1.07 to 103.66 ± 4.19
|
BBM
|
1.71
|
39.07 ± 1.58
|
BBM to BBM4P
|
1.71 to 0.53
|
30.59 ± 2.12 to 31.58 ± 0.86
|
3
|
BBM &BBM1/2NP
|
1.71 to 1.47
|
80.72 ± 1.55 to 83.72 ± 2.53
|
BBM
1/2NP
|
1.47
|
42.96 ± 1.75
|
BBM
1/2NP
|
1.47
|
35.94 ± 1.04
|
Halochlorella rubescenes
|
1
|
BBM3N
|
5.13
|
105.04 ± 2
|
BBM
1/2N
|
0.85
|
30.57 ± 0.82
|
BBM2N
|
3.42
|
25.66 ± 0.11
|
2
|
BBM & BBM2P
|
1.71 to 0.98
|
82.2 ± 2.03
|
BBM to BBM4P
|
1.71 to 0.53
|
25.41 ± 1.63 to 26.95 ± 0.65
|
BBM
|
1.71
|
21.48 ± 1.05
|
3
|
BBM1/2NP & BBM2NP
|
1.95 & 1.47
|
86.79 ± 2.55 to 90.77 ± 2.54
|
BBM
1/2NP
|
1.47
|
30.96 ± 0.22
|
BBM
1/2NP
|
1.47
|
26.87 ± 0.84
|
Coccomyxa simplex
|
1
|
BBM1/2N
|
0.85
|
72.94 ± 2.61
|
BBM
1/2N
|
0.85
|
30 ± 1.34
|
BBM
1/2N
|
0.85
|
21.86 ± 0.68
|
2
|
BBM2P & BBM3P
|
0.68 to 0.98
|
71.1 ± 3.01 to 73.18 ± 3.02
|
BBM4P
|
0.65
|
27.89 ± 0.7
|
BBM
1/2P
|
2.72
|
19.04 ± 0.99
|
3
|
BBM1/2NP
|
1.95
|
70.18 ± 2.02
|
BBM
1/2NP
|
1.47
|
32.62 ± 0.52
|
BBM
1/2NP
|
1.47
|
22.9 ± 0.74
|
Chlorolobion
braunii
|
1
|
BBM3N & BBM4N
|
5.13 to 6.84
|
107.62 ± 8.02 to 109.23 ± 3.03
|
BBM
1/2N
|
0.85
|
34.32 ± 0.89
|
BBM
1/2N
|
0.85
|
34.14 ± 1.59
|
2
|
BBM1/2P
|
2.72
|
109.49 ± 4.82
|
BBM
1/2PtoBBM4P
|
2.72 to 0.53
|
26.15 ± 0.47 to 27.89 ± 0.45
|
BBM
1/2P to BBM
|
2.72 to 1.71
|
27.49 ± 0.44 to 28.64 ± 1.39
|
3
|
BBM
|
1.71
|
102.18 ± 2.02
|
BBM
1/2NP
|
1.47
|
32.96 ± 0.22
|
BBM
1/2NP
|
1.47
|
30.03 ± 1.01
|
Kirchneriella
obesa
|
1
|
BBM & BBM2N
|
1.71 to 3.42
|
86 ± 1 to 87.09 ± 2.01
|
BBM
1/2N
|
0.85
|
22.99 ± 1.16
|
BBM &BBM
1/2N
|
0.85 to 1.71
|
17.02 ± 0.55 to 17.13 ± 0.86
|
2
|
BBM
|
1.71
|
86 ± 1
|
BBM
4P
|
0.53
|
24.44 ± 0.66
|
BBM
|
1.71
|
17.13 ± 0.86
|
3
|
BBM & BBM2NP
|
1.71 to 1.47
|
84 ± 3.61 to
86 ± 1
|
BBM
1/2NP
|
1.47
|
25.76 ± 0.86
|
BBM & BBM
2NP
|
1.71 & 1.47
|
17.13 ± 0.86 & 17.47 ± 0.59
|
Table 3
The highest Nitrogen Removal Rate (NRR), Nitrogen Removal Efficiency (NRE) of the five species of algae
Algae
|
Exp
|
Treatment
|
N:P
|
Nitrogen Removal Rate (NRR)
|
Treatment
|
N:P
|
Nitrogen Removal Efficiency (NRE)
|
---|
Monoraphidium contortum
|
1
|
BBM3N
|
5.13
|
3.53 ± 0.19
|
BBM
1/2N to BBM
|
0.85 to 1.71
|
91.09 ± 3.58 to 94.4 ± 4.83
|
2
|
BBM1/2P to BBM4P
|
2.72 to 0.53
|
1.65 ± 0.01
to 1.86 ± 0.08
|
BBM1/2P to BBM
|
1.71
|
94.4 ± 4.23
|
3
|
BBM2NP
|
1.95
|
2.41 ± 0.74
|
BBM
|
1.71
|
94.4 ± 2.28
|
Halochlorella rubescenes
|
1
|
BBM2N &BBM3N
|
3.42 to 5.13
|
3.47 ± 0.07 to 3.87 ± 0.36
|
BBM
|
1.71
|
96.05 ± 0.19
|
2
|
BBM1/2P to BBM4P
|
2.72 to 0.53
|
1.88 to 1.99 ± 0.7
|
BBM1/2P to BBM3P
|
2.72 to 0.68
|
94.84 ± 0.12 to 96.34 ± 0.29
|
3
|
BBM2NP
|
1.95
|
3.08 ± 0.7
|
BBM
|
1.71
|
96.05 ± 0.19
|
Coccomyxa simplex
|
1
|
BBM3N
|
5.13
|
2.91 ± 0.11
|
BBM
|
1.71
|
96.05 ± 0.19
|
2
|
BBM1/2P to BBM3P
|
2.72 to 0.68
|
1.89 ± 0.01to 1.97 ± 0.8
|
BBM1/2P to BBM3P
|
2.72 to 0.68
|
94.84 ± 0.12 to 96.34 ± 0.29
|
3
|
BBM
|
1.71
|
1.99 ± 0.06a
|
BBM
|
1.71
|
96.05 ± 0.38
|
Chlorolobion
braunii
|
1
|
BBM2N
|
3.42
|
3.23 ± 0.26
|
BBM
|
1.71
|
95.85 ± 0.1
|
2
|
BBM1/2P to BBM4P
|
2.72 to 0.53
|
1.87 ± 0.3 to 2.02
|
BBM
1/2PtoBBM3P
|
2.72 to 0.68
|
95.08 ± 1.71 to 96.87 ± 0.91
|
3
|
BBM2NP
|
1.95
|
2.05 ± 0.4
|
BBM
1/2NP &BBM
|
1.47 to 1.71
|
93.45 ± 3.32 to 95.85 ± 0.1
|
Kirchneriella
obesa
|
1
|
BBM3N &BBM4N
|
5.13 to 6.84
|
2.31 ± 0.38 to 2.45 ± 0.62
|
BBM
1/2N to BBM
|
0.85 to 1.71
|
93.88 ± 1.48
|
2
|
BBM1/2P to BBM3P
|
2.72 to 0.68
|
1.74 ± 0.63 to 1.94 ± 0.06
|
BBM
|
1.71
|
93.3 ± 2.92
|
3
|
BBM2NP
|
1.95
|
2.28 ± 0.52
|
BBM
1/2NP to BBM
|
1.47 to 1.71
|
87.95 ± 5.47 to 93.3 ± 6.92
|
Table 4
The highest Phosphorus Removal Rate (PRR) and phosphorus Removal Efficiency (PRE) of the five species of algae
Algae
|
Exp
|
Treatment
|
N:P
|
PhosphorusRemoval Rate (PRR)
|
Treatment
|
N:P
|
Phosphorus Removal Efficiency (PRE)
|
---|
Monoraphidium contortum
|
1
|
BBM1/2N
|
0.85
|
2.67 ± 0.15
|
BBM
1/2N
|
0.85
|
79.33 ± 4.35
|
2
|
BBM2P
|
0.98
|
2.45 ± 0.15
|
BBM1/2P
|
0.85
|
82.74 ± 0.7
|
3
|
BBM2NP
|
1.95
|
3.57 ± 0.11
|
BBM2NP
|
1.95
|
84.89 ± 2.63
|
Halochlorella rubescenes
|
1
|
BBM1/2N
|
0.85
|
2.21 ± 0.18
|
BBM
1/2N to BBM4N
|
0.85 to 6.84
|
66.35 ± 2.75 to 68.66 ± 5.34
|
2
|
BBMto BBM4P
|
1.71 to 0.53
|
1.94 ± 0.13
|
BBM1/2P
|
0.85
|
80.9 ± 0.12
|
3
|
BBM
|
1.71
|
1.94 ± 0.13
|
BBM
1/2NP
|
1.47
|
74.31 ± 0.68
|
Coccomyxa simplex
|
1
|
BBM1/2N
|
0.85
|
1.93 ± 0.05
|
BBM
1/2N
|
0.85
|
63.99 ± 1.41
|
2
|
BBM to BBM4P
|
1.71 to 0.53
|
0.79 ± 0.51 to 1.03 ± 0.09
|
BBM1/2P toBBM
|
0.85 to 1.71
|
37.51 ± 5.53 to 33.53 ± 3.35
|
3
|
BBM2NP
|
1.95
|
0.63 ± 0.07
|
BBM
|
1.71
|
37.51 ± 0.53
|
Chlorolobion
braunii
|
1
|
BBM1/2N
|
0.85
|
2.57 ± 0.23
|
BBM1/2N
|
0.85
|
76.25 ± 4.78
|
2
|
BBM2P to BBM4P
|
0.98 to 0.53
|
2.46 ± 0.07 to 2.6 ± 0.04
|
BBM
1/2PtoBBM
|
0.85 to 1.71
|
71.84 ± 5.4 to 75.39 ± 2.59
|
3
|
BBM
|
1.71
|
2.15 ± 0.31
|
BBM
1
|
1.71
|
71.84 ± 10.4
|
Kirchneriella
obesa
|
1
|
BBM
1/2N to BBM
|
0.85 to 1.71
|
1.97 ± 0.1 to 2.05 ± 0.16
|
BBM
1/2N to BBM4N
|
0.85 to 6.84
|
57.29 ± 1.06 to 68.72 ± 5.43
|
2
|
BBM2P to BBM4P
|
0.98 to 0.53
|
2.37 ± 0.21 to 2.83 ± 0.25
|
BBM1/2P
|
0.85
|
94.19 ± 0.75
|
3
|
BBM1/2NP to BBM
|
1.95 to 1.71
|
1.95 ± 0.05 to 2.05 ± 0.16
|
BBM
1/2NP
|
1.95
|
95.84 ± 0.56
|
Biomass productivity at varying N and P regimes in BBM
The details of biomass productivity of the five different species at varying treatments of the three experiments are given in Supplementary Table 1. The regular BBM was the control in all three experiments. Figure 2a, b, and c demonstrate the responses of the five species to the diverse treatments in the three experiments.
Figure 2a shows the biomass productivity of the five species at increasing N:P from half the level to four times N in BBM, causing an increase in N:P in BBM. All the algae showed unique responses in biomass productivity to the varying N regimes in BBM. In general, the biomass productivity of these species at varying N regimes in BBM was significantly different from each other except for Chlorolobion braunii and Halochlorella rubescens at specific treatments (Supplementary Table 1). The alga Coccomyxa simplex showed its highest biomass productivity at half the level of N, whereas Kirchneriella obesa showed its lowest at this level. However, in both of them, biomass productivity remained the same as that of the control at all other treatments. Halochlorella rubescens showed a significant increase in productivity compared to the control, up to three times N in BBM. In contrast, Monoraphidium contortum showed a similar increase in productivity, up to only two times N in BBM. Chlorolobion braunii showed a significant increase in its productivity from the control at 3 to 4 times N in BBM.
Figure 2b demonstrates the biomass productivity of the five species from half the BBM level to four times P (KH2PO4) in BBM, causing a decrease in N:P in BBM. In general, responses in productivity of the five algae to P variations in BBM were also unique. While Chlorolobion braunii and Coccomyxa simplex significantly increased biomass productivity compared to control at half the level of P in the BBM, all the other species significantly decreased biomass productivity at the same treatment.
In Chlorolobion braunii, the productivity decreased significantly from control up to 2 times P in BBM but remained constant afterward. In Coccomyxa simplex, the biomass productivity remained the same as that of control till three times P and then went significantly lower than control at four times P in BBM. In Halochlorella rubiscens, biomass productivity was significantly lower than control from two to 4 times P in BBM. Kirchneriella obesa biomass productivity was significantly lower than control in all the consecutive higher P treatments. However, biomass productivity of Monoraphidium contortum increased significantly from half the level of P to 2 times P in BBM and remained the same up to 4 times P in BBM. Thus, it became clear that Monoraphidium contortum is a productive green alga suitable for P-rich media with an N:P lower than BBM's.
Figure 2c shows the biomass productivity of the five species at two varied N and P together treatments in BBM while N:P remains constant. Like the other two experiments, all species' biomass productivity was unique at varying amounts of N and P, keeping N:P the same in the medium. While Kirchneriella obesa and Chlorolobion braunii had their productivity significantly lower than the control at half the level of N and P together in BBM, the others had their productivity same as that of the control at this treatment. In the second treatment of N and P together at two times that of BBM, the productivity of Kirchneriella obesa was the same as that of control, whereas that of Halochlorella rubescens was significantly higher than that of control. However, productivity was substantially lower in all the other three species than the control at this second treatment.
Overall, the biomass productivity of Chlorolobion braunii (highest productivity of 109.23mg l-1 day-1 at 3-4N in BBM) stands significantly higher than all the other algae at all the tested concentrations of N in BBM, revealing it as a productive green alga suitable for N-rich growth media with a broad increase in N:P (0.85 to 6.84). On the other hand, the biomass productivity of Monoraphidium contortum (highest productivity of 103.66mg l-1 day-1 at 2-4P in BBM) stands significantly higher than the other algae at high concentrations of P in BBM, revealing it is a productive species suitable for P-rich growth media with a narrow range of low N:P (0.53 to 0.98). However, the biomass productivity of Halochlorella rubescens (highest productivity of 90.77mg l-1 day-1) stands significantly higher than all others at two times N and P in BBM (N:P = that of BBM).
In general, green algal growth and yield are related to the total amount of N or P in the medium [36], and a low N:P ratio (lower than that of BBM) is considered unfavorable for the growth of green algae [37]. In the current experimental study, low N:P could be created by increasing P in the BBM or decreasing N in the BBM while keeping P constant. Accordingly, in the current study, Monoraphidium contortum showed significantly higher biomass productivity at a low N:P (0.98 to 0.53) by increasing P in the medium (2–4 times KH2PO4 in the BBM). The P content in BBM is 0.053 g/L, and BBM with 2–4 times P (KH2PO4) in the medium is 0.093 to 0.173 gL-1. In the current study, Monoraphidium contortum showed a significant increase in biomass productivity and a rise in P in medium from 0.093 gL-1 to 0.173 gL-1.
In contrast, its biomass productivity decreased significantly along with a decrease in P content at 0.053 gL-1 (BBM level) to 0.033 gL-1 (half the P in BBM) in the medium. Dhup et al. (2017) [38] observed a decline in the growth of Monoraphidium contortum at 0.1 gL-1 P. In contrast, in the current study, a decrease in productivity was observed for the species at 0.053 gL-1 P only. However, an increase in growth of the species at P content at 0.093 (two times KH2PO4 in BBM) for this species is found while keeping N at the BBM level in the medium. Moreover, the species showed no significant difference in productivity at higher N and P together in BBM. Thus, it became clear that P in the medium positively influences this species' growth. Therefore, Monoraphidium contortum may be suitable for treating P-rich waste waters. However, the optimum P regime needs to be standardized through further experimentation.
Overall, the biomass productivity of all five algae was unique in the BBM and showed individual responses to variations in N:P concerning N and P. Therefore, it may be concluded that specific N:P and the amount of N or P in the medium are decisive to the productivity of each algal species in a growth media. The primary significance of the current experiment is that it enabled simultaneous identification of the specific influence of N:P and N and P content on the growth and productivity of five species by adding varying amounts of N or P and N and P together into growth media. Thus, the current study emerged as a model experiment to identify the optimum nutrient requirement of indigenous species and to check whether they are suitable for p-rich or N-rich wastewaters.
The positive response of algae to low N in the medium is attributed to a lack of initial lag phase [39] in the growth cycle of such species of algae. In the present experiment with five indigenous species, Coccomyxa simplex is identified as such a species. In laboratory culture studies, limiting light availability for individual cells may affect growth above a critical culture density [40]. However, the difference in cell size of specific algal species determines their specific limit to light from high population density in the cultures. Accordingly, in the present experiment, light limitation on biomass productivity was observed for large cell-sized Halochlorella rubescens at three times N in the medium. In contrast, light limitation on biomass productivity of small cell-sized Chlorolobion braunii did not arise up to 4 times N in the growth medium.
According to Li et al. (2008) [40], sodium nitrate concentrations above ten mM in the media are toxic to green microalgae. Our experiments have shown that all algae except Halochlorella rubescens and Monoraphidium contortum are nitrate-tolerant species capable of tolerating Sodium nitrate content up to about 12 mM in the media. After all, their productivity remained the same at 3 to 4 times N in the BBM. In contrast, Halochlorella rubescens and Monoraphidium contortum are observed as nitrate-sensitive species because their growth or biomass productivity at three times N was significantly lower than that at two times N (10 mM) in the growth media. Dhup and Dhawan (2014) [41] also observed the Nitrate sensitivity of Monoraphidium spp. in their experiment. A decrease in the growth and productivity of algae at high Nitrate content in growth media can be due to an increase in nitrate reductase activity, which enhances the production of nitrite and ammonia that are accumulated in vivo [42].
According to [36], an N:P above five or more is favorable for the growth of microalga Coelastrum morus. In contrast, in the current study, a higher N:P, up to 6.84, is advantageous for Chlorolobion braunii. In comparison, P-varied treatments enabled us to show that Monoraphidium contortum and Coccomyxa simplex have the potential to grow at shallow N:P (0.98). Thus, The current experiments helped us explain how N:P concerning N or P in the growth media is significant to specific algal biomass productivity in the growth media, which is essential to utilize them for the phycoremediation of specific industrial wastewaters.
The productivity of Chlorolobion braunii (109.23 ± 3.03 mg l-1 day-1 at four-times N in BBM), Halochlorella rubescens (105.04 ± 2 mg l-1 day-1 at three-times N in BBM) and Monoraphidium contortum (103 ± 2.01mg l-1 d-1 at two to four-times P in BBM) discovered in the current study is higher than that of some other indigenous productive species of green microalgae previously reported from eutrophic waters of Kerala [25, 30].
Lipid yield and productivity
One of the most significant goals of the current investigation was to assess the lipid yield and productivity of the five species of algae under varied nutrient regimes of N and P concerning N:P ratios in the BBM. The details of the lipid yield and lipid productivity of the five species in all three experiments and the varied treatments are shown in Supplementary Table 2. Figure 3 and Fig. 4 depict specific trends in lipid yield and lipid productivity of the five species, respectively, concerning variations in N, P, and N:P. Since lipid productivity is the product of biomass productivity and lipid yield, it also varies in species per biomass productivity. Accordingly, the lipid productivity of all the species was unique, with significant differences between them at all the treatments.
In the first experiment of N-varied treatments, all five species showed significantly higher lipid yield at the first treatment of half the level of N in BBM than the control and all the other treatments. The exciting trend was decreased lipid yield and increased N or N:P. However, the highest lipid productivity among all the five species of algae was shown by Chlorolobion braunii at half the N level (0.85 N:P) in BBM. The other species showed their specific productivity lower than that of Chlorolobion brauinii at all the other treatments. Kirchneriella obesa showed the lowest productivity among all species at all the treatments.
In the second experiment of P-varied treatments, the five algal species showed unique lipid yield responses to each tested level of P in the BBM. Among all the species and all the treatments, Monoraphidium contortum showed the highest lipid yield at the control. In contrast, Kirchineriella obesa showed the lowest yield at two times P in BBM. In the P-varied experimental treatments, all the algae showed lipid productivity per each algae's biomass productivity. Accordingly, Monoraphidium contortum showed the highest lipid productivity at the half level of P, two times P, and four times P in BBM without any significant difference in lipid productivity between these treatments. However, Kircheriella obesa showed the lowest lipid productivity at four times P in BBM.
In the third experiment of N and P together treatments in BBM, all five species showed significantly higher lipid yield in the first treatment of half the level of N and P in BBM than the control and at all other treatments.
As in the previous experiments of individual N treatments, all the species showed their highest lipid yield at half the N and P together treatments. In this experimental treatment, Monoraphidium contortum showed significantly higher lipid yield and productivity than all other species at half N and P in BBM. Similarly, Kircheneriella obesa showed the lowest lipid yield at two times N and P treatment. However, the lowest lipid productivity among all species over the different treatments was shown by Cocomyxa simplex at two times N and P in the BBM. Overall, Monoraphidium contortum emerged as a green alga with the highest lipid yield and productivity at a lower nutrient regime (N or P or N and P together) than the BBM.
The details of lipid productivity for all five species in various treatments of all three experiments are presented in Fig. 4. Indigenous microalgae with efficient photosynthetic systems are well known for their rapid growth rate and lipid yield compared to traditional plants [43]. Such microalgae with consistent growth and strong adaptability are to be carefully screened for their protein and lipids content in their commercial applications [44], especially for phycoremediation cum biomass production. Lipid productivity is the product of biomass productivity and lipid yield; it depends on the biomass productivity of algae. A decreasing trend of lipid content with an increase in N in the current study was similar to the observation of [40]. Usually, lipid synthesis within algae increases as an adaptation to environmental stress, such as lack of nutrients [45]. Moreover, reducing nitrogen levels can stimulate lipid production in algae because nitrogen scarcity leads to the buildup of carbon precursors in the form of acetyl-CoA, which serves as a fundamental building block for synthesizing lipids [46]. Additionally, it is well-known that low N content induces a decline in algal growth and a shift in cell metabolism, mainly towards lipid accumulation, which can produce more lipids in some algae species [26].
Unlike the positive influence of a reduction of N on lipid productivity, a decrease in P in the medium didn't stimulate lipid productivity in any of the studied species in the current study. However, P deprivation induces excessive lipid content in some algae [47, 48]. On the contrary, in the present study, some species showed a significant increase in lipid content at four times P in the medium than at regular BBM. While excessive P in the medium may act negatively on biomass productivity, the cell metabolism in such species shifts towards lipid production.
It may be noted that Monoraphidium contortum, Halochlorella rubescens, and Coccomyxa simplex showed their highest lipid content and lipid productivity at half N and P together in BBM treatment. It agrees with the observation of [38] that a decrease in P and N below the optimum level can boost biomass productivity because of better nutrient utilization. It is well known that under nutrient stress, microalgal cells accumulate more cellular lipids in cells, up to 50 to 70% of the biomass, with neutral lipids (TAGs) making up the most significant proportion of this accumulation [49].
Another factor influencing lipid productivity is the N:P ratio of the medium. All the three species mentioned above showed their highest lipid productivity at N:P of 1.4, similar to the optimal nutrient regime for achieving the highest lipid yield in Chlorella surukiana [44]. However, N and P content and proportions may vary significantly for various microalgae species [50]. In the current study, different microalgae species also showed variations in maximum lipid productivity at varied N:P.
Fatty acid profile of the five species
The fatty acid profile of all five species of algae was carried out at their highest lipid productivity. The details of the same are presented in Table 5. The fatty acids of the five species of algae studied range from C8:0 to C24:0. The predominant saturated fatty acids are palmitic acid (C16: 0) and stearic acid (C18: 0) in these species. However, oleic acid (C18:1) was these algae's primary monounsaturated fatty acid. The major polyunsaturated fatty acids in these microalgae were linoleic and (C18:2) and linolenic acid (C18:3). The fatty acids such as C8:0, C15:0, C20:0, C21:0, C22:0, C24:0, C16:1, C18:3n6, C20:5n3 were present only in some of the selected species (Table 5).
Table 5
Fatty acid profile (% of total FAME) of five algae at its highest lipid-productivity
Fatty Acid
|
Monoraphidium contortum
|
Halochlorella rubescenes
|
Coccomyxa simplex
|
Chlorolobion
braunii
|
Kirchneriella
obesa
|
---|
C8:0
|
ND
|
0.138528
|
ND
|
0.014857
|
ND
|
C12:0
|
0.179802
|
0.190476
|
0.541272
|
0.148566
|
0.125122
|
C14:0
|
0.988912
|
0.519481
|
1.623816
|
0.282276
|
0.569999
|
C15:0
|
0.25472
|
ND
|
0.383401
|
0.074283
|
0.375365
|
C16:0
|
27.58466
|
45.78355
|
32.00271
|
23.78547
|
24.09287
|
C17:0
|
0.269703
|
0.380952
|
0.473613
|
ND
|
0.236341
|
C18:0
|
4.225352
|
2.233766
|
2.796572
|
2.124499
|
2.113166
|
C20:0
|
ND
|
0.363636
|
0.270636
|
ND
|
ND
|
C21:0
|
0.25472
|
ND
|
ND
|
ND
|
ND
|
C22:0
|
0.149835
|
ND
|
ND
|
0.267419
|
0.597803
|
C24:0
|
0.674258
|
ND
|
1.082544
|
0.267419
|
ND
|
∑% SFA
|
34.582
|
49.61
|
39.175
|
26.965
|
28.111
|
C16:1
|
0.869044
|
0.225108
|
ND
|
ND
|
5.755596
|
C18:1n9t
|
41.62421
|
25.78355
|
30.35634
|
50.79483
|
45.04379
|
C20:1
|
ND
|
0.658009
|
0.721696
|
1.188531
|
1.014876
|
C22:1n9
|
0.25472
|
0.900433
|
1.195309
| |
0.125122
|
∑% MUFA
|
42.748
|
27.567
|
32.273
|
51.983
|
51.939
|
C18:2n6c
|
11.13275
|
13.43723
|
9.855661
|
7.829446
|
8.563882
|
C18:3n3
|
11.01289
|
9.264069
|
18.49346
|
13.2224
|
11.20534
|
C18:3n6
|
0.344621
|
0.121212
|
0.202977
|
ND
|
0.180731
|
C20:5n3
|
0.179802
|
ND
|
ND
|
ND
|
ND
|
∑% PUFA
|
22.67
|
22.823
|
28.552
|
21.052
|
19.95
|
n-6
|
11.47737
|
13.55844
|
10.05864
|
7.829446
|
8.744613
|
n-3
|
11.19269
|
9.264069
|
18.49346
|
13.2224
|
11.20534
|
n-6/n-3
|
1.025435
|
1.463551
|
0.543902
|
0.592135
|
0.780397
|
ND not detected |
The total saturated fatty acid (SFA) ranged from 26.96 (Chlorolobion braunii at half the N in BBM) to 49.61% (Halochlorella rubescens at half the N &P in BBM). The monounsaturated fatty acid (MUFA) content ranged from 27.567% (Halochlorella rubescens at half the N & P in BBM) to 51.983% (Chlorolobion braunii at half the N in BBM). The polyunsaturated fatty acids (PUFA) in these algae ranged from 19.95 (Kirchneriella obesa at 4 N in BBM) to 28.55% (Coccomyxa simplex at half the N &P in BBM). Therefore, among the tested microalgal species, Chlorolobion braunii (50.79%), Kirchneriella obesa (45.04%), and Monoraphidium contortum (41.62%) showed the highest oleic acid content, making it the most suitable for the production of good quality biodiesel.
Biodiesel properties of the algal lipids
In evaluating the caliber of the five microalgae species for their biodiesel quality, empirical equations were used to estimate the same from their respective fatty acid profile (Table 6). All five species studied showed the Cetane Number (CN) ranging from 51.31 to 55.65 and matched the American standard for biodiesel biodiesel (ASTM D6751) and European biodiesel standards (EN14214).
Table 6
Fuel properties of biodiesel produced from the five green microalgal species at its highest lipid-productivity
Sl No
|
Biodiesel quality parameter
|
Monoraphidium contortum
|
Halochlorella rubescenes
|
Coccomyxa simplex
|
Chlorolobion
braunii
|
Kirchneriella
obesa
|
---|
1
|
IV (g I2 100g− 1)
|
90.268
|
74.784
|
97.983
|
97.018
|
93.868
|
2
|
SV (mg KOH g− 1)
|
204.668
|
208.48
|
206.039
|
203.386
|
204.915
|
3
|
CN
|
52.657
|
55.654
|
50.744
|
51.307
|
51.815
|
4
|
DU (wt%)
|
88.088
|
73.212
|
89.378
|
94.087
|
91.839
|
5
|
HHV (MJ kg− 1)
|
40.424
|
40.338
|
40.362
|
40.449
|
40.409
|
6
|
OS (h)
|
7.792
|
7.758
|
6.721
|
8.192
|
8.502
|
7
|
LCSF (wt%)
|
6.954
|
6.059
|
7.034
|
4.377
|
4.363
|
8
|
CFPP (°C)
|
5.37
|
2.558
|
5.623
|
-2.727
|
-2.771
|
9
|
CP (°C)
|
9.518
|
19.09
|
11.841
|
7.519
|
7.681
|
10
|
PP (°C)
|
3.511
|
13.902
|
6.034
|
1.342
|
1.517
|
IV iodine value, SV saponification value, CN cetane number, DU degree of unsaturation, HHV higher heating value, OS oxidative stability, LCSF long-chain saturation factor, CFPP cold filter plugging point, CP cloud point, PP pour point.
Iodine value (IV), which measures the total unsaturation within a mixture of fatty acids, is another crucial data. The calculated IV for five species ranged from 74.78 to 97.983; all showed < 120 that matched EN14214. All five microalgal species had higher heating value (HHV), within the predetermined range (40.26 to 42.65 MJ kg− 1) for ordinary biodiesel, which is typically 7–12% less than that of diesel made from petroleum (46 MJ kg− 1).
For fuel applications requiring low temperatures, the cloud point (CP), pour point (PP), and cold filter plugging point (CFPP) are crucial. While the PP is the temperature at which the wax-formed fuel can flow, the CP is the temperature at which a cloud of wax crystals initially forms when the fuel is cooled. All species except Halochlorella rubescens (CP = 19.09°C) exhibited CP values ranging between 7.52 to 11.841°C and PP values 1.342 to 13.902°C, matching the ASTM D6751 standard. The CFPP values ranged from − 2.727 to 5.62°C, demonstrating the suitability of biodiesel for all five algae at low temperatures.
The EN 14214 set the oxidative stability as anything above six h. All species recorded 6.72 to 8.502 h oxidative stability in the present case. The saponification value, which measures the average molecular weight of all the fatty acids in algal lipids, ranged from 203.386 to 208.48. In the present study, the Degree of Unsaturation (DU) value ranges from 73.212 to 94.09%, making it suitable for long-term storage.
The fatty acid profile is an essential criterion for selecting a microalgal species for phycoremediation cum biomass utilization as a nutraceutical or biofuel resource [51]. Chlorophyceaen algal species predominantly have C16:0, C18:0, C18:1n9c, and C18:2n6 fatty acids [26, 52]. The unsaturated fatty acid content generally makes microalgae extremely stress-responsive to flourish in nutrient-deficient environments. Unsaturated fatty acid content also enables them to have improved membrane fluidity for thriving under adverse conditions [53].
In general, an ideal balance of saturated and unsaturated FAMEs in cells determines the good biodiesel quality of microalgae. The primary fatty acids determining the fuel qualities are palmitic, oleic, and linoleic acids [54], present in the desired proportion in all the studied algae. Higher concentrations of C16 and C18 fatty acids are advantageous for biodiesel quality [55], and the findings of this study met the requirement for high-quality biodiesel. Usually, vegetable oils with a high oleic acid content have a decent fuel balance, including ignition quality, combustion heat, cold filter plugging point (CFPP), oxidative stability, viscosity, and lubricity [56]. The highest oleic acid content was found in Chlorolobion braunii (50.79%), followed by Kirchneriella obesa (45.04%), and the least oleic acid was found in Halochlorella rubiscens (25.78%)
Additionally, the fatty acid composition of algal oil determines biodiesel quality, such as oxidation stability, cetane rating, melting point, viscosity, and fluidity at low temperatures. The low level of polyunsaturated fatty acids and a large number of saturated fatty acids act as crucial markers of biodiesel quality because unsaturated bonds decrease the oxidative stability of biodiesel [57]. However, oils with high unsaturated fatty acid content and a low melting point are preferred in biodiesel production for low-temperature uses [58]. The CN is a frequently used indicator for determining fuel quality because it is decisive in the combustion behaviour of the fuel's readiness to self-ignite when injected [35]. The minimal CN levels for biodiesel are 47.0 and 51.0, respectively, per ASTM D6751 and EN14214 standards. Therefore, the lipids of all five algae with above 51 CN satisfy such criteria for biofuels. However, Halochlorella rubescens did not meet the CP limit of -3 to 12 set by EN 14214 among the five algae tested.
The NRR, PRR, NRE, and PRE of the five microalgae
The details of the NRR and NRE of all five algae are given in Supplementary Table 3, whereas PRR and PRE are given in Supplementary Table 4. The trends in their responses concerning NRR are shown in Fig. 5a, b, and c, whereas PRR is demonstrated in Fig. 6a, b, and c, respectively. Overall, the NRR, PRR, NRE, and PRE of the five species of green algae concerning variations in N or P and N and P together enabled us to check the potentials of five indigenous green algae in response to different regimes of N, P concerning N:P in the growth medium. The details of the best NRE, NRR, PRE, and PRR concerning N or P or N and P together, and N:P in BBM by the five species are shown in Tables 3 and 4, respectively.
The NRE and NRR of the five microalgae
In the first experimental treatments of variations of N in BBM, keeping P constant, all five species showed their highest NRE at the control (93.3 to 96.05%) without any significant difference among them. At half the N in the BBM (N:P = 0.85), the NRE was significantly lower than the control in Halochlorella rubescens. The NRE of all other species remained the same as that of the control at half the level of N in the medium. However, in all species, at all levels of increase of N in the BBM (N:P 1.71 to 6.84), the NRE remained lower than that of the control. In the second experiment of varying P keeping N constant in the BBM, the NRE of all species at half the P in BBM (N:P = 2.72) remained the same as that of the control. However, on increasing P in the BBM (N:P 1.71 to 0.53), the NRE of all the species remained the same or decreased only. In the third experiment, at half the level of N and P together in BBM (N:P = 1.71), the NRE of the three species significantly decreased from that of the control. However, in Chlorolobion braunii and Kirchneriella obesa, the NRE remained the same as that of the control during this treatment. The NRE of all species showed a significant decrease from that of the control at two times N and P in BBM (N:P = 1.71).
In general, in all species, the NRR at half the N in BBM was significantly lower than that at the control (Fig. 5a). On increasing N in the BBM, Halochlorella rubiscens, Monoraphidium contortum and Cocomyxa simplex showed a significant increase in NRR from the control (regular BBM) up to 3 times N in BBM (N:P 1.71 to 5.13); but at 4N (N:P = 6.84), the NRR decreased significantly from that of the control (Fig. 5a). In Chlorolobion braunii the NRR increased significantly from the control up to 2N (N:P = 3.42), and it remained the same at 3N. However, its NRR significantly decreased at 4N, like the first three species. In Kirchneriella obesa, NRR remained the same at control and 2N but increased significantly at 3N but remained the same on further increase up to 4N. Overall, the highest NRR was exhibited by Halochlorella rubescens (3.87mg l-1 day-1) and Monoraphidium contortum (3.53 mg l-1day-1) at 123.48 ppm N without any significant difference in NRR by the two species.
In the second experimental condition of varying P in the regular BBM (Fig. 5b) keeping N constant, except Coccomyxa simplex and Kirchneriella obesa, all the other three species showed the same NRR at all variations of P in BBM. However, in Coccomyxa simplex and Kirchneriella obesa, NRR remained the same up to 3 times P in BBM, significantly decreasing at four times P in the medium.
In the third experiment of varying N and P together in BBM without a change in N:P, the NRR of all species at half the N and P in BBM was significantly lower than that of the control. At an increase of NP up to two times the level of BBM (Fig. 5c), the NRR of all algae except Coccomyxa simplex and Chlorolobion braunii significantly increased from that of the control. In Coccomyxa simplex, NRR decreased significantly, whereas in Chlorolobion braunii, NRR remained the same without any significant difference from control in two times N and P in BBM.
All species demonstrated the highest NRE at the control and half the N in BBM without any significant difference between the treatments. Moreover, at a constant level of phosphorus (P), NRR and NRE of the five tested species varied based on the nitrogen (N) content and nitrogen-to-phosphorus (N:P) ratio in the medium.
It is well-known that at low nitrogen content in medium, NRE is almost complete [59]. This is because microalgae utilize both nitrogen and phosphorus together for their growth. Naturally, an excessive supply of one major nutrient element exhausts the other essential nutrients from the growth medium, leading to no further change in NRE [60].
The significance of N:P on algal growth and nutrient removal was first proposed by Redfield (1958) [61], and the optimal N:P for marine phytoplankton per Redfield is 16 mol-N/mol-P. Subsequent studies reveal that the optimal N:P of specific microalgae varies according to the species and may also be influenced by the particular conditions of cultivation depending on their adaptation to culture conditions [62]. Although N:P has no influence on NRR by certain species [36], the present study revealed that an increase in N:P positively influences the NRR of microalgae, and the effect is species-specific.
In the present study, the optimum N:P for the maximum NRR was 5.13 in Halochlorella rubiscens, Monoraphidium contortum, and Cocomyxa simplex. However, the optimum N:P for Chlorolobion braunii was 3.42, and for Kirchneriella obesa, 6.84. The N:P ratio significantly impacts nutrient usage by microalgae, the optimum of which depends on the species [63]. However, according to Dhup and Dhawan (2014) [41], the highest nutrient uptake rate is associated with an N:P ratio of 0.153 for certain algae.
PRE and PRR of the five microalgae
In N treatments, Monoraphidium contortum, Chlorolobion braunii, and Coccomyxa simplex showed significantly higher PRE at half the level of N in the BBM than at all other treatments. Halochlorella rubescens and Kirchineriella obesa PRE were the same as the control at half the level of N in BBM. However, towards an increase of N in the BBM, the PRE remained the same as that of the control. In general, Monoraphidium contortum (with PRE 62–79%) and Chlorolobion braunii (with PRE 66–76%) are found to be the best among the five species for removal of P from N-rich media. In P treatments, Monoraphidium contortum, Halochlorella rubescens, and Kirchneriella obesa showed significantly higher PRE at half the level of P in the BBM than all other treatments. In contrast, the PRE of Chlorolobion braunii and Coccomyxa simplex remained equal to that of control. However, in all species, the PRE remained lower than that of the control towards an increase in P in BBM.
In N and P together treatments, the PRE of the three species, Monoraphidium contortum, Chlorolobion braunii, and Coccomyxa simplex, was significantly lower than that of the control, whereas that of Halochlorella rubescens and Kirchneriella obesa remained same as that of control. The PRE of all species except Monoraphidium contortum showed a significant decrease from that of the control at two times N and P in BBM. Monoraphidium contortum showed a significantly higher PRE than the control at two times N and P together in BBM.
In the N treatments, all the species except Kirchneriella obesa showed significantly higher PRR than the control at half the level of N in BBM. In contrast, Kirchneriella obesa remained the same as that of control. However, on increasing N in the BBM, the PRR responses of all the species remained the same as that of the control or decreased only (Fig. 6a).
In the P treatments, the different species showed a unique increase in PRR. The algae Halochlorella rubescens and Kirchneriella obesa showed a rise in PRR to 3P in BBM. In contrast, Monoraphidium contortum and Chlorolobion braunii showed a surge in PRR up to 2 P in BBM. The PRR of Coccomyxa simplex remained the same as that of control without any significant difference up to 4 times P in BBM (Fig. 6b). It may be noted that each species is suited best for growth media of different phosphorus regimes. Chlorolobion braunii has its best PRR (2.5 mg l-1 day-1 PRR at 0.98 N:P), Kirchneriella obesa (2.83 mg l-1 day-1 at 0.68 N:P) and Monoraphidium contortum (2.45 mg l-1 day-1 at 0.98 N:P).
In the N and P together treatment, PRR decreased significantly for all the species from the control in the first treatment of half the level of N and P in BBM; however, on increasing N and P together to two times, PRR either remained the same (Kirchneriella obesa) or decreased (Coccomyxa simplex, Chlorolobion braunii & Halochlorella rubescens). However, in Monoraphidium contortum, PRR increased exponentially to 3.57 mg l-1 day-1 at the third treatment (N:P = 1.71) (Fig. 6c).
Among the five species, only Kirchneriella obesa showed increased PRR and a decrease in N in the medium. A similar response is known earlier [41] in Monoraphidium contortum. [38] PRE decreases with increased P for all microalgae species, but the tolerance limit is species-specific. According to the author, an elevated phosphorus content usually harms the culture, hindering algal growth because of inefficient resource utilization. The nutrient removal efficiency declines because the cells avoid uptake beyond a certain threshold. In the present study, PRR increased along with P in the medium, but the response was species-specific. A similar demonstration of an increase in PRR, along with an increase in phosphorus in the media, is known [36].