Toxic effects of surfactants on grazer growth and reproduction
The successive transfer cultures of the two grazers (Poterioochromonas sp. and Hemiurosomoida sp.) were established first as described in Methods. Using these successive transfer cultures, the efficacies of the five selected surfactants for controlling Poterioochromonas sp. and Hemiurosomoida sp. were evaluated. Toxic effects on both Poterioochromonas sp. and Hemiurosomoida sp. were observed for all five surfactants, namely SDBS, CDEA, SDS, AEO-7, and AES.
As shown in Fig. 1, a greater than 30% increase in cell densities of Poterioochromonas sp. were obtained after 24 h cultivation without surfactant addition, suggesting the viability of the Poterioochromonas sp. cultures. However, the cell densities decreased in the cultures supplemented with any one of the five surfactants. For example, the cell density of the living Poterioochromonas sp. was 2.8 × 104 cells mL− 1 in the culture without SDBS addition, yet it decreased to 1.8 × 104 cells mL− 1 in the culture supplemented with 3 mg L− 1 SDBS and further decreased to less than 100 cells mL− 1 with 6 mg L− 1 SDBS treatment. No living Poterioochromonas sp. were observed microscopically when the SDBS concentration was further increased to 8 mg L− 1, which we considered as the complete control of Poterioochromonas sp. contamination.
The decreasing trend in Poterioochromonas sp. density with increasing surfactant concentration was found for all five tested surfactants, suggesting that they affect the grazer Poterioochromonas sp. similarly, however, for each surfactant, the minimal effective concentrations to completely control the contamination were different. SDBS and AEO7 were the most powerful reagents, eliminating Poterioochromonas sp. completely at concentrations not lower than 8 mg L− 1. Second, the efficacies of CDEA and SDS on Poterioochromonas sp. were similar and their minimal effective concentrations were 10 and 12 mg L− 1, respectively. AES showed weak efficacy on controlling of Poterioochromonas sp., with a minimal effective concentration of 20 mg L− 1.
Toxic effects of the five surfactants on Hemiurosomoida sp. were also observed (Fig. 2). The viability of Hemiurosomoida sp. was shown by an increased in cell densities, which were more than 40% higher in comparison to the initial density in the culture without surfactant supplementation. Hemiurosomoida sp. densities decreased significantly after surfactants addition. Taking the SDBS treatment as an example, almost 60% decrease in the Hemiurosomoida sp. density, from 1.6 × 103 cells mL− 1 to 680 cells mL− 1, was obtained when 4 mg L− 1 SDBS was supplemented into the culture. A further increase in the SDBS concentration (10 mg L− 1) led to the complete elimination of Hemiurosomoida sp. and no living cells were observed under the microscope. The general trends of decreasing cell densities with increasing surfactant concentrations were also detected for the five surfactants. However, the efficacies against Hemiurosomoida sp. were not the same as that for Poterioochromonas sp. The most powerful one was AEO7, which eliminated Hemiurosomoida sp. at a concentration of 8 mg L− 1. The next ones were SDBS and CDEA, the minimal effective concentrations of which were 10 mg L− 1 and 15 mg L− 1, respectively. The complete elimination of Hemiurosomoida sp. by AES was only obtained at 30 mg L− 1. A substantial difference was observed in SDS, which had a minimal effective concentration of 12 mg L− 1 for Poterioochromonas sp., but at least 35 mg L− 1 SDS was needed to completely eliminate Hemiurosomoida sp.
Effects of the five surfactants on Chlorella growth
Chlorella pyrenoidosa XQ-20044 was cultured under different concentrations of the five surfactants to evaluate the surfactant effects on cell growth, photosynthetic activity, and viability. Data of the SDBS exposure experiment are shown in Fig. 3 as an example; other data concerning CDEA, SDS, AES, and AEO7 are provided in Additional file 1.
The time courses of the Chlorella biomass DW showed no significant difference when the SDBS concentration was less than 20 mg L− 1 (Fig. 3a). The biomass DW of the culture having no SDBS supplementation reached 0.72 g L− 1 on day 3, with an average growth rate of 0.84 d− 1. Smaller but insignificant biomass DW (0.67 g L− 1) and growth rate (0.82 d− 1) were obtained in the culture with 20 mg L− 1 SDBS supplementation. However, the biomass DW was only 0.41 g L− 1 with a significantly decreased growth rate of 0.66 d− 1 when the SDBS concentration was further increased to 40 mg L− 1.
The photosynthetic activity of Chlorella (Fig. 3b) showed that in comparison to the SDBS-free culture, the changes in the photochemical yield of Chlorella cells were very small after 3 days of exposure to 20 mg L− 1 SDBS. The ratio between variable fluorescence and maximum fluorescence (FV/FM) of Chlorella was 0.72 in the SDBS-treated (20 mg L− 1) culture in the present study. This value fell into the general FV/FM range of dark-adapted green microalgae [17], suggesting that the photosynthetic activity of C. pyrenoidosa XQ-20044 was not influenced by SDBS at concentrations lower than 20 mg L− 1.
FDA staining (Fig. 3b, 3c) clearly showed membrane integrity and viability of the Chlorella cells, with similar fluorescein fluorescence intensities in both the SDBS-treated (20 mg L− 1) and the contrast culture. All of the above results suggested that Chlorella biomass yield may be reduced due to over exposure to SDBS, but the influences of SDBS was negligible at a concentration not higher than 20 mg L− 1.
Application of sodium dodecyl benzene sulfonate (SDBS) as a pesticide to control flagellates and ciliates grazing on Chlorella in raceway pond
The SDBS surfactant was further tested outdoors to validate the laboratory data. According to its performance in the raceway ponds, more technical details with respect to its outdoor application are discussed.
According to our observation, naturally occurring contaminations of Poterioochromonas sp. or Hemiurosomoida sp. can be observed generally on days 2–4 of a newly inoculated Chlorella culture in an outdoor raceway pond (unpublished results). This trend was successfully mimicked by the addition of Poterioochromonas sp. or Hemiurosomoida sp. “seeds” into the Chlorella culture ponds (Fig. 4). 18S rDNA based metagenomic data for identification of the contaminating species can be seen in Additional file 2. As soon as continued increases in grazer densities were observed for 3–4 days, for example, the grazer Hemiurosomoida sp. increased continually from 1.0 × 105 cells L− 1 on the 4th day to 2.7 × 105 cells L− 1 on the 5th day and 6.4 × 105 cells L− 1 on the 6th day, and further increased to 1.4 × 106 cells L− 1 the next day, the cultures were treated with 10 mg L− 1 SDBS to control Hemiurosomoida sp. or Poterioochromonas sp., and the other parallel cultures allowed contaminations to develop.
As shown in Fig. 4, cell densities of the grazers Poterioochromonas sp. and Hemiurosomoida sp., increased regularly for 3 or 4 days. The target microalgae C. pyrenoidosa XQ-20044 also showed a quick increase in cell density (indicated by Chl a content) during this period because the grazer populations were not large enough to have a significant grazing effect on Chlorella. The increase in grazer densities continued thereafter in the cultivations without SDBS addition, with the majority of grazers swallowing plenty of Chlorella cells and enclosing in their bodies. When the densities of Poterioochromonas sp. and Hemiurosomoida sp. reached approximately 3.6 × 107 cells L− 1 and 6.4 × 105 cells L− 1, respectively, the Chlorella density decreased due to grazing. By comparison, almost all the Poterioochromonas sp. and Hemiurosomoida sp. cells disintegrated and disappeared in one day in the cultivations with SDBS addition (10 mg L− 1) on the 6th day and 7th day, respectively, with the Chlorella growth kept as normal.
Overall, the final Chlorella biomass concentration reached 0.6 g L− 1 after a 12-day cultivation applying SDBS pesticide. It was only 0.26 g L− 1 if the Poterioochromonas-contamination was not controlled and 0.17 g L− 1 if the Hemiurosomoida-contamination was not controlled (Fig. 5). These data suggest that by applying 10 mg L− 1 SDBS as a pesticide to control Poterioochromonas sp. or Hemiurosomoida sp. contamination, the reduction in Chlorella biomass yield, which was estimated to be greater than 60% owning to grazer contamination, can be avoided. Actually, economic loss caused by biological contamination was much bigger than expected because the residual Chlorella biomass could only be used as low-quality raw materials when no effective steps were taken to manage the contaminations. The working concentration of SDBS (10 mg L− 1) was slightly higher than the minimal effective concentration to eliminate Poterioochromonas sp. in the laboratory. This was to simplify the application that using one uniform concentration to control both Poterioochromonas and Hemiurosomoida contaminations.
SDBS pesticide was also applied in 20 and 200 m2 cascade cultures of Chlorella at October 2019 (Fig. 6). Two rounds of contamination naturally occurred during the process, both of which were Poterioochromonas sp. contaminations. The first round of Poterioochromonas contamination was observed early on the 2nd day in the 20 m2 pond. The cell density of Poterioochromonas increased gradually from 7.6 × 104 cells L− 1 to 1.1 × 106 cells L− 1 on the 3rd day, and then drastically increased over the following days with densities on the 4th and 5th days reaching 8.1 × 106 cells L− 1 and 2.8 × 107 cells L− 1, respectively. During this time, the Chlorella density was not significantly influenced because the grazer density was relatively low. SDBS addition (10 mg L− 1) on the 5th day resulted in a sharp decrease in Poterioochromonas sp. density and the grazer was rarely observed over the following days. The alga could still grow and continuously increase its biomass owning to the robust effects of the SDBS pesticide. A cell density of 11.1 mg Chl a L− 1 (0.42 g DW L− 1, alternatively 8.4 g m− 2 d− 1) was observed on the 10th day. The biomass yield of Chlorella was comparable to those previously reported [18, 19]. On the 10th day, the culture was scaled up into a 200 m2 raceway pond and four days later the second round of Poterioochromonas contamination was observed. The development of the second round of contamination was very similar to the previous one observed in the 20 m2 pond. SDBS pesticide (10 mg L− 1) successfully eliminated Poterioochromonas sp. once again, without damaging Chlorella growth.