Qiang Penga,
Miaomiao Zhaob,
Guangzhu Shenb,
Xinyu Ganb and
Ming Li*bcd
aCollege of Food Science, Northwest Agriculture and Forestry University, Yangling 712100, PR China
bCollege of Resources and Environment, Northwest Agriculture and Forestry University, Yangling 712100, PR China. E-mail: lileaf@163.com
cKey Laboratory of Plant Nutrition and the Agri-environment in Northwest China, Ministry of Agriculture, PR China
dCollaborative Innovation Center of Water Security for Water Source Region of Mid-line of South-to-North Diversion Project, Henan Province, PR China
First published on 30th January 2017
The lipids found in microalgae are promising raw materials for bio-diesel production. This study tests the effects of linear alkylbenzene sulfonate (LAS) on the growth, colony formation, sedimentation and lipid content of the green alga Scenedesmus obliquus. Growth of S. obliquus was unaffected by LAS below concentrations of 100 mg L−1. LAS treatment promoted colony formation and the number of cells per aggregate significantly increased with rising LAS concentration. Both optic and scanning electron microphotoes showed that the induced colony of S. obliquus was regular and there were clear attachment points between two neighboring cells. The sedimentation efficiency at 10 min significantly increased with the increasing LAS concentration. Under treatment of 100 mg L−1 LAS, the sedimentation efficiency was 69.0% after 40 minutes' settling, which was 30% higher than that in the control group. The sedimentation efficiency was positively related to cells per colony at all sedimentation time except for 60 min. Moreover, the lipid content was 21.2% when treated with 100 mg L−1 LAS, which was 1.18 times higher than in the control group. The main fatty acid composition of S. obliquus was α-linolenic acid (C18:3) and palmitic acid (C16:0). The degree of fatty acid unsaturation was not obviously affected by LAS addition but the proportion of fatty acid with the chain length ranging from C16 to C18 was promoted from 70.9% to 84.2% by LAS addition. This result indicated that LAS addition was beneficial to improve the quality of algal biodiesel. Our results suggested that moderate doses of LAS can effectively enhance the sedimentation efficiency of S. obliquus by inducing colony formation and improving the lipid content of the cells without inhibiting their growth.
Algal biomasses are frequently harvested by adding synthetic polymer flocculants and metal coagulants, which stimulate single cells to form large aggregates.4 Generally, this method cannot be widely applied because synthetic polymer flocculants are expensive and metal coagulants are toxic to algal cells.5 Alkaline flocculation is also an alternative method for algae harvest. However, the sedimentation efficiency of alkaline flocculation was always inhibited by the extracellular polymeric substances (EPS) produced by algal cells even though addition of minim alkali is cheap.4 Therefore, a cheap and effective chemical that promotes algal biomass harvesting is imperative.
In an alternative method, Yang and Li6 stimulated colony formation of unicellular S. obliquus by adding Daphnia culture filtrates. The filtrates were considered to significantly increase the colony size of S. obliquus and hence improve the harvesting efficiency.7 Nevertheless, large Daphnia culture filtrates themselves consume much energy and demand significant financial resources. Low light intensity was also reported to induce colony formation of S. obliquus8 whereas algal biomass growth would also be inhibited due to low light intensity. Therefore, the gains of this method cannot compensate the losses.
Li et al.9 demonstrated that 100 and 1000 mg L−1 linear alkylbenzene sulfonate (LAS) induced colony formation of S. obliquus. This phenomenon revealed that LAS would promote sedimentation of S. obliquus by inducing colony formation. Meanwhile, LAS is a widely used representative surfactant.9 It is cheap, and easily manufactured and degraded. As a consequence, LAS is considered as a promising additive for promoting colony formation and sedimentation of S. obliquus. However, Li et al.9 studied the effects of LAS on colony formation of S. obliquus and found two defects limiting the development of technology to promote sedimentation of S. obliquus by LAS addition. On one hand, the concentration gradient of LAS used in their work was too big to analyze the appropriate concentration of LAS.9 On the other hand, the authors only investigated colony size of S. obliquus rather than the sedimentation efficiency.9 Hitherto, there was not a clear finding illustrating the relationship between colony size and sedimentation efficiency of S. obliquus even in the work of Zhu et al.,7 in which combined effects of nitrogen levels and Daphnia culture filtrate on colony size of S. obliquus were well studied. Therefore, it is still important to study the effects of LAS on colony size and sedimentation efficiency of S. obliquus with proper concentration gradient of LAS.
The algal fatty acids composition was another important factor affecting algal biodiesel production.10,11 The increase in the degree of fatty acid unsaturation was in favor of high-quality biodiesel production because this production was more suitable for cold weather use due to a typically gel point.12 Moreover, the chain length of fatty acids are also selective and the most optimum chain length was ranging from C16 to C18.10,11 Our knowledge about the effects of LAS on algal fatty acid composition was seriously inadequate. Therefore, the effects of LAS addition on algal fatty acid composition should also be evaluated.
This study tests the effects of LAS addition on colony formation and sedimentation efficiency of S. obliquus. As lipid productivity and fatty acid composition are two important reference standards in algal diesel production,13 the growth, lipid content and fatty acid composition of LAS-stimulated S. obliquus were also analyzed.
Under the same culture conditions, the algae in logarithmic-phase were inoculated in 150 mL BG-11 medium in 250 mL conical flasks with an initial cell density of 5 × 105 cells per mL. Various amount of LAS were added to the culture medium, and their concentrations were adjusted to 0, 25, 50, 100, 200 and 500 mg L−1. LAS with a chemical formula of C18H29NaO3S was obtained from Sigma Chemical Company (USA). Six replicates of each culture were prepared. The flasks were hand-shaken two to three times daily to prevent the cells from clinging to the inner walls of the flasks. Colony formation of algae always occurred in the logarithmic growth phase14 but lipid content in the stable phase was much higher than that in the logarithmic growth phase.15 Gan et al.16 showed that the growth of S. obliquus entered the stable phase for day 16 and thus the experiment ran for 14 days in the current study as a compromise.
Sedimentation efficiency (%) = (1 − ODt/ODi) × 100 | (1) |
μ = ln(Dt/D0)/t | (2) |
![]() | ||
Fig. 3 Photos of harvested biomass as well as optical and scanning electron microphotoes (SEM) of S. obliquus. |
The relationship between sedimentation efficiency at varying sedimentation time and cells per particle of S. obliquus was shown in Fig. 5. The sedimentation efficiency was positively related to cells per colony at all the sedimentation time except for 60 min. If cells per particle reached 8, sedimentation efficiency reached 60% at 10 min and the value was much higher than 70% at 20 min.
![]() | ||
Fig. 5 The relationship between sedimentation efficiency and cells per particle of S. obliquus at varying sedimentation time. |
![]() | ||
Fig. 6 Lipid contents of S. obliquus treated with various concentrations of LAS. Different lowercase letters indicate significant differences (P < 0.05) among treatments. |
The main fatty acid composition of S. obliquus was α-linolenic acid (C18:3) and palmitic acid (C16:0) showing in Table 1. The proportion of α-linolenic acid (C18:3) was not affected by LAS addition except for the treatment with highest level, in which the value decreased from 45.5% to 32.2% when treated with 500 mg L−1 LAS. On the contrary, the proportion of palmitic acid (C16:0) increased from 16.3% to 23.7% with increasing LAS concentration. The degree of fatty acid unsaturation was not obviously affected by LAS addition. However, the proportion of fatty acid with the chain length ranging from C16 to C18 was promoted from 70.9% to more than 76% (highest value reached 84.2%) by LAS addition.
Fatty acid | Concentration of LAS in the treatment (mg L−1) | |||||
---|---|---|---|---|---|---|
0 | 20 | 50 | 100 | 200 | 500 | |
C15:0 | 0 | 3.3 | 3.8 | 0 | 0 | 0 |
C16:0 | 16.3 | 22.6 | 18.9 | 20.0 | 22.0 | 23.7 |
C18:0 | 0 | 1.8 | 1.6 | 0 | 0 | 0 |
C18:1 | 0 | 8.6 | 9.1 | 8.6 | 7.7 | 22.5 |
C18:2 | 9.1 | 3.5 | 4.6 | 0 | 4.7 | 4.9 |
C18:3 | 45.5 | 47.7 | 46.7 | 47.4 | 46.3 | 32.2 |
C20:5 | 16.2 | 10.4 | 13.7 | 16.2 | 13.5 | 11.7 |
C22:0 | 12.9 | 2.1 | 1.6 | 7.8 | 5.8 | 5.0 |
Unsaturated | 70.8 | 70.2 | 74.1 | 72.2 | 72.2 | 71.3 |
C16–C18 | 70.9 | 84.2 | 80.9 | 76.0 | 80.7 | 83.3 |
It was also found that sedimentation efficiency at 10, 20, 30, 40 and 50 min was significantly related to cells per particle of S. obliquus. These regression analysis results could be used to predict the sedimentation performance of S. obliquus based on a large number research results about the effects of environmental factors on colony size of S. obliquus.7,8 These predictions would provide some references to choose proper agent to promote sedimentation of S. obliquus for harvesting.
Our results also illustrated that colony formation occurred in all the treatment with varying concentrations of LAS from 25 mg L−1 to 500 mg L−1. Li et al.9 reported that 100 mg L−1 LAS successfully induced colony formation of S. obliquus but 10 mg L−1 LAS did not succeed in inducing colony formation. Here, we obtained the missing information that 25 and 50 mg L−1 LAS can also induce colony formation. Colony formation in S. obliquus is thought to be triggered by increased EPS production.8,21 LAS can inhibit protein and DNA synthesis in S. obliquus, and reduce its dark respiration activity.22 Thus, the reduced polysaccharide consumption could increase the EPS content and hence induce colony formation. This mechanism has been proved previously by Li et al.9 There were clear attachment points between two neighboring cells of induced colony of S. obliquus. Frame and Sawa23 showed that single cells of S. obliquus were wrapped by a structureless slimy layer. Therefore, the pathway of colony formation in the current study was that cells of S. obliquus remain attached after binary fission due to the wrapping of slimy layer.
In the current study, the proportion of fatty acid with the chain length ranging from C16 to C18 was promoted by LAS addition. That is to say LAS addition benefitted to improve the quality of algal biodiesel.10,11 The algal fatty acid composition, related to algal physiological state, was usually affected by salinity.12,24 LAS addition also increased salinity and thus affected fatty acid composition in the current study. However, the effects of increasing alkyl benzene sulfonic acid on algal fatty acid composition was still not clear and need further studies.
Compared with control, 25 mg L−1 LAS significantly stimulated lipid accumulation in S. obliquus. Merely, there was no significant change in the lipid content after addition of 50–500 mg L−1 LAS. Similar phenomenon that lipid content increased with increasing NaCl when NaCl concentration was low but decreased slightly when NaCl concentration was high was also reported by Salama et al.12 Wang et al.25 reported that low concentrations of LAS enhanced the maximal photochemical efficiency of Microcystis aeruginosa. It could also been inferred that low concentration of LAS might also enhance the maximal photochemical efficiency of S. obliquus improving its light harvesting for lipid synthesis and thus promoted the accumulation of lipid. When LAS concentration was high, photosynthesis of S. obliquus was inhibited22 and thus no significant change in the lipid content was found in the treatment of 50–500 mg L−1 LAS. As is well documented, inorganic substances such as nutrients,26 Fe,27 CO2 (ref. 2) and salt12 can promote lipid accumulation in S. obliquus. Glucose addition also reportedly increases the lipid content,26 because some S. obliquus strains are facultative heterotrophs.28 However, the lipid content of S. obliquus did not monotonically increase with LAS concentration, indicating that LAS is not an organic carbon source for S. obliquus in the current study. The effects of Na+ can also be excluded, because the maximum Na+ concentration in the present treatments was 39.9 mg L−1, too low to exert any significant effect. Additionally, the levels of triacylglycerol (TAG) and other small molecules (such as glycerol) increase under changes in osmotic pressure,29 which could be induced by surfactant addition. These molecules promote lipid accumulation in the algal cells.12
Although the 25 mg L−1 LAS treatment significantly stimulated lipid accumulation in S. obliquus (Fig. 6), its effect on sedimentation efficiency was not superior. In contrast, 100 mg L−1 LAS maximized the sedimentation efficiency at 40 minutes (to >30% above the control group), but yielded moderate lipid content (1.18 times the control lipid content). The filtrates can be recycled to reduce costs. Lürling30 reported that surfactant (FFD-6) induces colony formation of S. obliquus within 24 h. Therefore, the LAS concentration in the culture medium could be adjusted to 25 mg L−1 using recycled filtrate, then adjusted to 100 mg L−1 by LAS addition one day before harvesting. The first step would boost the cell yield and the second would enhance the lipid content.
This journal is © The Royal Society of Chemistry 2017 |