Toxic effects of surfactants on predator growth and reproduction
The successive transfer cultures of the two predators (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 sodium dodecyl benzene sulfonate (SDBS), coconut diethanolamide (CDEA), sodium dodecyl sulfate (SDS), fatty alcohol polyoxyethylene ether (AEO-7), and alcohol ethoxysulphate (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, demonstrating 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 predator Poterioochromonas sp. similarly. However, for each surfactant, the minimal effective concentrations to completely control the contamination were different. SDBS and AEO-7 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 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 AEO-7, 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 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 AEO-7 are provided in Additional file 1.
The time courses of the Chlorella biomass DW showed no significant difference (P > 0.05) 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 (P > 0.05) 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 (P < 0.05) 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, 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. 3c, d) 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 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 above results demonstrate that SDBS and AEO-7 were powerful surfactants for controlling Poterioochromonas and Hemiurosomoida contaminants while had little effect on Chlorella growth. Considering that SDBS is more easily degraded in natural environment and is less toxic to aquatic organisms than AEO-7 [23], SDBS was further tested outdoors to validate the laboratory data. 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. The predator densities increased continuously for 3–4 days after inoculation. For example, Hemiurosomoida sp. increased from 1.0 × 105 cells L−1 on the 4th day to 1.4 × 106 cells L−1 on the 7th day. At this time the cultures were treated with 10 mg L−1 SDBS, and contaminations in other parallel cultures were not treated and allowed to develop.
As shown in Fig. 4, cell densities of the predators Poterioochromonas sp. and Hemiurosomoida sp. increased consistently 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 predator populations were not large enough to have a significant grazing effect on Chlorella. The increase in predator densities continued thereafter in the cultivations without SDBS addition. When the densities of Poterioochromonas sp. and Hemiurosomoida sp. reached approximately 3.6 × 107 and 6.4 × 105 cells L−1, respectively, the Chlorella density decreased due to predation. By comparison, almost all the Poterioochromonas sp. and Hemiurosomoida sp. cells disintegrated and disappeared in one day in the cultivations after 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 as 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% owing to predation, 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). The cascade cultures were initiated in a 20-m2 raceway pond. After 10 days of cultivation the culture suspension was used as seed for a larger culture in a 200-m2 raceway pond. 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 to 1.1 × 106 cells L−1 on the 3rd day, and then drastically increased with densities on the 4th and 5th days reaching 8.1 × 106 and 2.8 × 107 cells L−1, respectively. During this time, the Chlorella density was not significantly influenced because the predator 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 predator was not observed over the following days. The algal biomass increased continuously after addition 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 [24, 25]. On the 10th day, the culture was scaled up into a 200-m2 raceway pond and 4 days later the second round of Poterioochromonas contamination was observed. 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.