Keywords: Chlorella; metal nanoparticles; microalgae; lipid; biomass;
The presence of metallic nanomaterials can alter the inhibitory effect of metals on microalgae. This work had a positive approach of using metal nanoparticles to improve the microalgal growth under two phases. In the first phase, microalgae were cultivated in the presence of varying concentrations of metal nanoparticles to induce resistance against metals. The effect of metal nanoparticles and its effect in terms of specific growth rate and biomass concentration were determined. In the second phase, microalgae that has recorded significant increase in growth rate and biomass grown under metal nanoparticles was selected for further cultivation in the presence of metal salts. Both the metal nanoparticle exposed strain (MNPS) and wild strain (WS) were cultivated separately in metal salts containing medium. Growth rate, biomass concentration, pigment concentration, contents of protein, carbohydrate and lipid of two groups (MNPS and WS) were compared to determine the role of metal nanoparticles in deriving high value products from microalgae.
Varying concentrations of metal nanoparticles were added into Bristol medium and initial growth in the presence of metal nanoparticles was varying with different days of cultivation. The growth rate was increasing in the beginning followed by decline phase in most cases. The visible growth of microalgae was observed after 8 days in the presence of copper nanoparticles (CuNP), 13 days of cultivation period in the medium containing magnesium nanoparticles (MgNP), 7 days in lead nanoparticles (PbNP) and 11 days in zinc nanoparticles (ZnNP). Total cultivation period of 8 days from the initial growth day was considered for all the metal nanoparticles tested. Highest specific growth rate and biomass were observed in 20 mg L-1, 100 mg L-1, 50 mg L-1 and 50 mg L-1 concentrations for CuNP, PbNP, MgNP and ZnNP respectively during the cultivation period (Figure 1a-4b). Both the specific growth rate and biomass concentration were decreased with increasing days of cultivation in media containing CuNP, PbNP and MgNP whereas increasing trend was observed in the presence of ZnNP with longer cultivation period. Control experiments were carried out using Bristol medium without metal nanoparticles under the same cultivation conditions. In general, higher concentrations of metal nanoparticles induced growth inhibition of microalgae under the experimental conditions. In the case of zinc nanoparticles containing media, metal concentrations above 100 mg L-1 has inhibited the growth of microalgae up to 17 days of cultivation period. The difference in the growth of microalgae in the presence of various metal nanoparticles is due to type of metals used (Cu, Mg, Pb, Zn) and size of the nanoparticles (76 nm - 92 nm). A number of environmental factors are known to influence nanoparticle behaviour including pH, ionic strength and particle concentration [24-26].
In the second phase of the experiment, microalgae which exhibited maximum specific growth rate and biomass concentration was selected and used for further cultivation. This is based on the fact that the presence of metal nanoparticles had induced metal resistance in microalgae which in turn was able to survive and produce highest specific growth rate and biomass. The second phase involved the comparison of biochemical attributes of metal nanoparticle induced metal resistance microalgae with non-metal nanoparticle exposed microalgae. For this study, both the strains were cultivated for a period of 8 days in the presence of metal salts and the concentration is similar to that of metal nanoparticles.
Growth rate, biomass and biochemical attributes of tested microalgae groups grown in the presence of metal salts were evaluated (Table 1-4). In most cases, inhibitory effects of metal nanoparticles on microalgae were reported. Toxicity of silver nanoparticles and nickel oxide nanoparticles on microalgae were reported earlier [27, 28]. The effect of zinc nanoparticles on Chlorella sp was studied by [29] (Chen et al., 2012). The presence of metal nanoparticles has affected the photosynthetic pigments of Chlorella vulgaris [30]. Higher concentrations of Manganese oxide nanoparticles had inhibitory effect on Chlorella pyrenoidosa [31]. Toxicity of Zinc oxide nanoparticles on microalgae was studied by Pendashte et al., [32] and found that Chlorella species was more sensitive than Scenedesmus.
Cu NP |
WS |
MNPS |
|||||||||
Control |
10 mg |
20 mg |
30 mg |
40 mg |
50 mg |
10 mg |
20 mg |
30 mg |
40 mg |
50 mg |
|
SGR (µ d-1) |
1.25 ± 0.06 |
0.86 ±0.11 |
0.97 ± 0.10 |
0.91 ±0.04 |
0.82 ±0.11 |
0.80 ±0.21 |
0.91 ±0.04 |
0.99 ± 0.17 |
1.01 ±0.21 |
0.90 ±0.31 |
0.87 ±0.41 |
Biomass (g L-1) |
1.37 ± 0.20 |
0.94 ± 0.12 |
1.02 ± 0.71 |
1.09 ± 0.31 |
0.91 ± 0.07 |
0.86 ± 0.04 |
1.00 ± 0.51 |
1.11 ± 0.06 |
0.99 ± 0.35 |
0.92 ± 0.30 |
0.89 ± 0.09 |
Chlorophyll (mg L-1) |
2.83 ± 0.01 |
0.72 ± 0.63 |
0.52 ± 0.21 |
0.36 ± 0.12 |
0.29 ± 0.00 |
0.36 ± 0.05 |
1.36 ± 0.11 |
1.14 ± 0.21 |
0.96 ± 0.05 |
0.87 ± 0.02 |
0.78 ± 0.07 |
Carotenoid (mg L-1) |
1.79 ± 0.16 |
0.47 ± 0.06 |
0.36 ± 0.03 |
0.25 ± 0.11 |
0.24 ± 0.21 |
0.20 ± 0.02 |
0.38 ± 0.16 |
0.39 ± 0.08 |
0.43 ± 0.10 |
0.51 ± 0.23 |
0.43 ± 0.19 |
Protein (mg L-1) |
50 ± 0.17 |
23 ± 0.30 |
20 ± 0.35 |
11 ± 0.22 |
10 ± 0.14 |
9 ± 0.03 |
20 ± 0.29 |
22 ± 0.74 |
20 ± 0.79 |
25 ± 0.35 |
25 ± 0.15 |
Carbohydrate (mg L-1) |
16 ± 0.74 |
10 ± 0.09 |
14 ± 0.38 |
21 ± 0.61 |
18 ± 0.70 |
7 ± 0.34 |
30 ± 0.68 |
26 ± 0.41 |
20 ± 0.05 |
14 ± 0.36 |
10 ± 0.71 |
Lipid (mg L-1 d-1) |
0.15 ± 0.10 |
0.13 ± 0.09 |
0.19 ± 0.10 |
0.18 ± 0.01 |
0.11 ± 0.13 |
0.11 ± 0.92 |
0.19 ± 0.01 |
0.28 ± 0.01 |
0.20 ± 0.33 |
0.13 ± 0.87 |
0.12 ± 0.00 |
Mg NP |
WS |
MNPS |
|||||||
Control |
50 mg |
100 mg |
150 mg |
200 mg |
50 mg |
100 mg |
150 mg |
200 mg |
|
SGR (µ d-1) |
1.25 ± 0.06 |
0.98 ± 0.14 |
0.81 ± 0.25 |
1.14 ± 0.21 |
1.02 ± 0.10 |
0.94 ± 0.08 |
0.97 ± 0.04 |
1.04 ± 0.15 |
0.85 ± 0.20 |
Biomass (g L-1) |
1.37 ± 0.20 |
0.82 ± 0.01 |
0.86 ± 0.44 |
1.01 ± 0.06 |
1.21 ± 0.15 |
0.89 ± 0.11 |
0.98 ± 0.34 |
1.25 ± 0.31 |
1.03 ± 0.15 |
Chlorophyll (mg L-1) |
2.83 ± 0.01 |
1.81 ± 0.25 |
1.45 ± 0.36 |
0.76 ± 0.44 |
0.88 ± 0.75 |
1.77 ± 0.06 |
1.81 ± 0.09 |
1.97 ± 0.10 |
1.93 ± 0.06 |
Carotenoid (mg L-1) |
1.79 ± 0.16 |
0.51 ± 0.27 |
0.69 ± 0.19 |
0.51 ± 0.09 |
0.64 ± 0.52 |
0.47 ± 0.11 |
0.51 ± 0.20 |
0.60 ± 0.31 |
0.69 ± 0.29 |
Protein (mg L-1) |
50 ± 0.17 |
50 ± 0.64 |
35 ± 0.71 |
20 ± 0.58 |
10 ± 0.09 |
35 ± 0.29 |
32 ± 0.47 |
29 ± 0.53 |
32 ± 0.68 |
Carbohydrate (mg L-1) |
16 ± 0.74 |
14 ± 0.54 |
18 ± 0.39 |
28 ± 0.66 |
12 ± 0.29 |
18 ± 0.05 |
15 ± 0.36 |
16 ± 0.64 |
25 ± 0.51 |
Lipid (mg L-1 d-1) |
0.15 ± 0.10 |
0.21 ± 0.01 |
0.22 ± 0.41 |
0.43 ± 0.06 |
0.28 ± 0.15 |
0.22 ± 0.13 |
0.23 ± 0.10 |
0.46 ± 0.16 |
0.31 ± 0.07 |
Lead NP |
WS |
MNPS |
|||||||
Control |
25 mg |
50 mg |
75 mg |
100 mg |
25 mg |
50 mg |
75 mg |
100 mg |
|
SGR (µ d-1) |
1.25 ± 0.06 |
0.85 ± 0.11 |
0.89 ± 0.04 |
0.92 ± 0.14 |
1.04 ± 0.07 |
0.97 ± 0.02 |
1.04 ± 0.12 |
1.21 ± 0.07 |
1.31 ± 0.10 |
Biomass (g L-1) |
1.37 ± 0.20 |
0.92 ± 0.13 |
0.97 ± 0.20 |
1.04 ± 0.12 |
1.16 ± 0.04 |
0.99 ± 0.17 |
1.02 ± 0.07 |
1.27 ± 0.10 |
1.41 ± 0.11 |
Chlorophyll (mg L-1) |
2.83 ± 0.01 |
1.28 ± 0.16 |
1.49 ± 0.71 |
1.65 ± 0.20 |
1.81 ± 0.36 |
1.39 ± 0.04 |
1.51 ± 0.26 |
1.59 ± 0.17 |
1.72 ± 0.41 |
Carotenoid (mg L-1) |
1.79 ± 0.16 |
0.30 ± 0.22 |
0.30 ± 0.72 |
0.38 ± 0.68 |
0.69 ± 0.14 |
0.34 ± 0.19 |
0.35 ± 0.24 |
0.38 ± 0.51 |
0.39 ± 0.35 |
Protein (mg L-1) |
50 ± 0.17 |
48.8 ± 0.24 |
48.5 ± 0.54 |
61.7 ± 0.42 |
87.5 ± 0.63 |
45.5 ± 0.49 |
49.6 ± 0.64 |
58.5 ± 0.74 |
65.3 ± 0.23 |
Carbohydrate (mg L-1) |
16 ± 0.74 |
23.7 ± 0.10 |
12.9 ± 0.23 |
26.4 ± 0.04 |
34.3 ± 0.51 |
19.8 ± 0.08 |
21.9 ± 0.41 |
20.4 ± 0.48 |
19.7 ± 0.64 |
Lipid (mg L-1 d-1) |
0.15 ± 0.10 |
0.26 ± 0.08 |
0.56 ± 0.70 |
0.65 ± 0.58 |
0.76 ± 0.62 |
0.30 ± 0.11 |
0.49 ± 0.27 |
0.55 ± 0.20 |
0.65 ± 0.04 |
Zinc NP |
WS |
MNPS |
|||||||
Control |
50 mg |
100 mg |
150 mg |
200 mg |
50 mg |
100 mg |
150 mg |
200 mg |
|
SGR (µ d-1) |
1.25 ± 0.06 |
1.04 ± 0.10 |
1.01 ± 0.24 |
1.21 ±0.03 |
1.06 ±0.21 |
0.94 ± 0.05 |
0.96 ± 0.11 |
0.89 ± 0.20 |
0.81 ± 0.06 |
Biomass (g L-1) |
1.37 ± 0.20 |
1.20 ± 0.10 |
1.17 ± 0.07 |
1.41 ± 0.31 |
1.11 ± 0.24 |
1.44 ± 0.10 |
1.40 ± 0.20 |
1.35 ± 0.20 |
1.27 ± 0.20 |
Chlorophyll (mg L-1) |
2.83 ± 0.01 |
1.73 ± 0.11 |
1.97 ± 0.03 |
1.60 ± 0.21 |
1.36 ± 0.66 |
1.04 ± 0.20 |
1.97 ± 0.09 |
1.59 ± 0.16 |
1.43 ± 0.57 |
Carotenoid (mg L-1) |
1.79 ± 0.16 |
080 ± 0.14 |
0.50 ± 0.07 |
0.84 ± 0.11 |
0.11 ± 0.01 |
0.22 ± 0.05 |
0.34 ± 0.10 |
0.67 ± 0.07 |
0.55 ± 0.04 |
Protein (mg L-1) |
50 ± 0.17 |
65 ± 0.09 |
72 ± 0.10 |
54 ± 0.61 |
41 ± 0.24 |
62 ± 0.81 |
42 ± 0.06 |
32 ± 0.47 |
60 ± 0.31 |
Carbohydrate (mg L-1) |
16 ± 0.74 |
14 ± 0.55 |
12 ± 0.26 |
9 ± 0.09 |
8 ± 0.13 |
20 ± 0.41 |
45 ± 0.38 |
46 ± 0.28 |
29 ± 0.21 |
Lipid (mg L-1 d-1) |
0.15 ± 0.10 |
0.51 ± 0.77 |
0.58 ± 0.72 |
0.54 ± 0.50 |
0.37 ± 0.42 |
0.68 ± 0.22 |
0.57 ± 0.49 |
0.74 ± 0.17 |
0.53 ± 0.17 |
Magnesium at a concentration of 150 mg L-1 has produced highest biomass and chlorophyll content but is lesser than control. Magnesium is one of the key elements required for chlorophyll synthesis [34] and higher magnesium concentration and MgSO4 nanoparticles were found to enhance the lipid accumulation in Chlorella vulgaris [35]. This is in accordance with the present study where lipid content was significantly increased in both the groups (0.46 and 0.43 mg L-1) than control.
Higher concentrations of zinc metal had influenced the growth rate and biomass concentration of C. vulgaris in the study, There were variations in cellular pigments and protein contents in which WS recorded of higher content than MNPS. Increase in soluble protein content is considered as an evidence of active defense mechanism to prevent algae cells from damaging by abiotic stress [36] and increase in total protein content of the cells exposed to metal salts was observed in this study. The carbohydrate content was greatly influenced in MNPS and recorded 46 mg L-1 which is 80% and 65% higher than WS and control. Soluble sugars are effective candidates for capturing reactive oxygen species (ROS) and scavenging the free radicals in microalgal cells exposed to environmental stresses [37]. It was noteworthy to observe that the total lipid content was 0.74 mg L-1 in MNPS whereas it was 0.54 and 0.15 mg L-1 in WS and control respectively.
The study revealed that nanometal induced resistant strain has produced higher growth rate, biomass, cellular pigment, protein, carbohydrate and lipid content than non metal exposed strain when grown in the presence of metal salts. The data obtained were similar in most of the nanometals tested but the results were different for lead nanoparticles. In other words, growth rate and biochemical attributes were higher in WS than MNPS. Protein content of 87.5 mg L-1 was observed in WS grown in lead containing medium which is highest than other metal salts and control. Another significant observation is the increase in lipid content of microalgae as 0.76 mg L-1 which is also higher than any other metal salts studied. It was also noted that the lead metal in the form of lead acetate salt had positive effect on biomass and lipid content of C. vulgaris. The feasibility of mass culture of microalgae for biodiesel production greatly depends on high biomass productivity and lipid yield [38].
The inhibitory effects of nanoparticles on microalgae were reported widely and reports which revealed the positive effect of nanoparticles on various microalgae are also available. Increase in microalgal pigments were observed when the microalgae were cultivated in the presence of metal nanoparticle solutions [39]. The addition of lower concentrations of copper nanocarboxylates (20 to 40 mg L-1) and selenium nanocarboxylates (0.07 to 0.2 mg L-1) had stimulated the growth of Chlorella and increase in biomass along with chlorophyll content of the microalgae [40]. Both neutral and total lipid contents were increased in Scenedesmus obliquus in the presence of carbon nanotubes, Fe2O3 nanoparticles and MgO nanoparticles [41]. Zero-valent iron nanoparticles were found to boost the growth of several green algae [42].
In conclusion, this study evaluated the use of metal nanoparticles to induce metal resistance in C. vulgaris thereby increasing the high value products such as biomass, cellular pigments and lipid from microalgae. Initial experiments demonstrated the metal resistance development through metal nanoparticles and further experiments confirmed the positive influence of metal nanoparticles to improve microalgal growth, biomass and lipid production when grown in the presence of metal salts.
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