Microalgae are potential biomass feedstock for proteins, pigments, pharmaceuticals and biofuels, owing to their high efficiency of solar energy utilization, fast growth rate and ability to accumulate a high quantity of lipid. Furthermore, microalgal cultivation could be coupled with CO2 capture from industrial flue gases and removal of nitrogen and phosphorous compounds during wastewater treatment. Sustained, large-scale, biomass production is a prerequisite for realizing these potentials of microalgae. Biological contamination is one of the limiting factors in the outdoor large-scale cultivation of microalgae. The high grazing capacity of zooplankton can inhibit the algal concentration, prolonging the culture period or resulting in aquaculture system breakdown in a few days. However, there are no effective control methods for biological contamination in large-scale cultivation of microalgae. In this paper, Brachionus plicatilis and Stylonychia mytilus were selected as the experimental objects, (1) studying the mechanism that rotifer released undefined chemicals to inhibit the microalgal growth besides predation and the components of secretion were analyzed; (2) discussing the application effects and economic feasibility of toosendanin and celangulin in the control of biological contamination, in order to provide theoretical and technical guidance for the detection and control of biological contamination in the large-scale cultivation of microalgae.
The main results are as follows:
- The effects of different concentrations of rotifer secretion on the growth of Chlorella sp. were studied. The results showed that the bacteria-free, rotifer culture filtrate (RCF) significantly decreased Chlorella sp. cell densities during the incubation, and the microalgal cell densities decreased with increasing RCF proportions (5%, 10%, 20% and 30%) in fresh F/2 media. The responses of Chlorella sp. at different starting cell densities (1.4×106 cells mL−1, 4.2×106 cells mL−1, 7.8×106 cells mL−1 and 18.0×106 cells mL−1) to 10% RCF were different. Increasing the initial cell density of Chlorella sp. would disperse the inhibitory chemical(s) present in 10% RCF over more cells, reducing their effect on each cell, but the overall inhibitory effect was not changed with time. The results confirmed that the action of the chemical(s) released by rotifers on microalgal cell growth was dependent on both the RCF concentration and the exposure time. They also demonstrate that ≥ 10% RCF significantly inhibited photosynthesis and respiration, and inhibition rate was positively correlated with the proportion of RCF in F/2 medium. Calculations based on the data indicated that the rotifer-derived chemical(s) released hourly from each rotifer inhibits growth by 45.5±3.2 microalgal cells in addition to the rotifer predation.
- With the aim of identifying the chemical nature and action mechanism of the inhibitor released from rotifers, the responses of Chlorella sp. to different components of rotifer secretion was studied. The results showed that the rotifer inhibition of Chlorella sp. growth could not be eliminated by boiling the RCF, indicating that the inhibitor was not likely a protein. The growth and photosynthesis of Chlorella sp. was not inhibited by the water-soluble RCF fraction, but they were significantly inhibited by the lipid-soluble RCF fraction, in a dose-dependent manner. Further, the lipid-soluble fraction decreased energy conservation and photosynthetic electron transport, which induced a severe decrease in PSII activity and a decrease in the net photosynthetic O2 evolution rate. Based on these physiological responses of Chlorella sp. cells, the lipid-soluble fraction rather than the protein or water-soluble fractions was determined to contain the responsible inhibitor. The inhibitor, we speculated, was probably free fatty acids or substances derived from the photooxidation of unsaturated fatty acids.
- In order to further analyze the inhibition mechanism of lipid-soluble fraction of RCF, the antioxidant system of Chlorella sp. under 10% and 30% RCF lipid-soluble inhibitors was studied. The results showed that under the lipid-soluble inhibitor of RCF (10%), the contents of superoxide anion (O2-), hydroxyl radical (·OH) and hydrogen peroxide (H2O2) were increased, which indicated that the Chlorella sp. cells treated by inhibitors were damaged by peroxidation. At the same time, the activities of superoxide dismutase (SOD), aseorbateperoxidase (APX) and peroxidase (POD) in the antioxidant system increased, the hydroxyl free radical removal capacity (HFRSC) and glutathione (GSH) increased, indicating that Chlorella sp. tried to decrease the accumulation of active oxygen by improving antioxidant capacity. However, the lipid-soluble inhibitor of RCF (30%) significantly inhibited the activities of SOD, O2-, ·OH and HFRSC. Although the activity of SOD increased, the content of O2- and ·OH also increased significantly. The high concentration lipid-soluble inhibitor of RCF made chlorella sp. produced a lot of active oxygen, causing oxidative stress, which leads to lipid peroxidation, cell structure destruction and growth inhibition.
- In order to identify an effective technique for reducing rotifer contamination, the mechanism of Brachionus plicatilis inhibiting Nannochloropsis oculata and the efficacy of the celangulin : toosendanin (CA:TSN) (1:9) combination for rotifer extermination were investigated using chlorophyll a fluorescence transient. The results showed that B. plicatilis could directly devour N. oculata cells and sharply reduce an algal density to very low levels. B. plicatilis also inhibited the activities of PSII reaction centers, acceptor side and donor side in surviving N. oculata cells, and led to the imbalance between photosynthetic light absorption and energy utilization, even oxidative stress. However, the CA:TSN (1:9) combination could control B. plicatilis, thereby preventing B. plicatilis from devouring N. oculata cells and protecting photosynthetic electron transport chain of the surviving N. oculata cells against rotifers damage. Meanwhile, the CA:TSN (1:9) combination did not affect the growth of N. oculata. Therefore, the botanical pesticide, the binary combination of CA:TSN (1:9), is a good candidate of botanical pesticide for controlling rotifer contamination.
- Another common biological contamination - Stylonychia mytilus was selected as the experimental object in this experiment, and the toxic effects of toosendanin and ammonium bicarbonate on Chlorella pyrenoidosa were compared. Toxicity tests showed that toosendaninand ammonium bicarbonate were highly toxic to S. mytilus, with 24 h lethal concentration 50% (LC50) values of 6.4 μg L−1, and 0.8 g L−1, respectively. The population density of S. mytilus decreased significantly when exposed to ≥ 2 μg L−1 toosendanin, or ≥ 0.4 g L−1 ammonium bicarbonate. In addition, the S. mytilus control effects of toosendaninand ammonium bicarbonate and their safety in C. pyrenoidosa were evaluated. It was found that ≤ 14 μg L−1 toosendanin had no obvious toxic influence on photosynthesis and growth of C. pyrenoidosa and even increased the final cell density, with the highest being 12.3% over that of untreated cultures, and effectively reduced the S. mytilus density. Ammonium bicarbonate is the most widely used optimization technique for controlling contamination, but it has limited ability to reduce S. mytilus. However, ≥ 0.8 g L−1 ammonium bicarbonate inhibited photosynthesis and growth of C. pyrenoidosa, causing a 5.1% reduction in cell density or even a complete crop failure. Based on its high toxicity to S. mytilus and its relative safety to C. pyrenoidosa, toosendanin was considered to be a good potential botanical pesticide for controlling S. mytilus contamination in microalgal mass cultivation.
Based on the above results, we speculated that the lipid-soluble RCF fraction was probably free fatty acids or substances derived from the photooxidation of unsaturated fatty acids. The fraction destroyed the photosynthetic electron transport chain, causing the imbalance between photosynthetic light absorption and energy utilization, electron leakage of photosynthetic electron transport chain. Under the condition, a lot of active oxygen was produced, causing oxidative stress. Severe damage to the plasma membranes would cause cell structure destruction and growth inhibition. However, toosendanin and celangulin can effectively control B. plicatilis contamination and S. mytilus contamination in the large-scale cultivation of microalgae, with highly economic feasibility. A better understanding of the inhibition of microalgae by rotifers may help adjust contamination control schemes and optimize the productivity of algal cultures. Besides, a low-cost, environmentally friendly, easy-to-apply, pesticide treatment to control B. plicatilis and S. mytiluscontamination would provide a theoretical and technical guidance for the contamination control of harmful organisms in the large-scale cultivation of microalgae.