Studies On The Rotifer Extermination During Microalgal Cultivation

Microalgae have great potential applications in varied fields such as the food, nutritionand health care. Microalgal resource development can produce large amounts ofhigh-additional-value substances that are in great demand now, and is also considered to bean ideal way for renewable energy production. However, the contaminations caused bybiological pollutants constrain the algal large-scale cultivation and seriously impede thedevelopment of microalgal resource. Microalgal growth is especially susceptible to thegrazing by zooplanktons, which can inhibit algal growth and reduce the algal concentrationto low levels within just a few days, resulting in heavy economic loss. For now, no effectiveapproaches are found to control the biological contaminations. Based on the biologicaldifferences between zooplankton and phytoplankton, this study screened the individualbotanical pesticide that had high toxicity on rotifers, and obtained the synergistic binarycombinations of the pesticides. The rotifer-control effects of individual botanical pesticideand the synergistic combinations, and their safety for algal growth were studied. Theeconomics, practical applicability, and insecticidal mechanism of botanical pesticides onexterminating rotifers were also discussed. This study is aimed to provide theoretical andtechnical directions to the control of biological pollutants in microalgal mass cultivation. Themain results were as follows:1. By determining the acute toxicity of four botanical pesticides (celangulin,azadirachtin, matrine and toosendanin) on the rotifer B. plicatilis, three botanical pesticides,celangulin, matrine and toosendanin, that were highly acute toxic to rotifers were screened,with24h LC50values of0.175mg/L,0.061mg/L and2.132×10-3mg/L, respectively.Sublethal concentrations of celangulin (≥0.110mg/L), matrine (≥0.050mg/L) andtoosendanin (≥0.191×10-3mg/L) showed highly chronic toxicity toward the rotiferreproduction and population growth, with significant decreases of the net reproduction,intrinsic rate and finite rate of increase, and shortened generation time. The inhibitory effects of celangulin, matrine and toosendanin on population growth of rotifers were significantlyconcentration-dependent and time-dependent. The longer the time or higher theconcentration the rotifers were exposed to, the stronger the population growth of rotifers wasinhibited.2. The rotifer-control effects of individual botanical pesticide and its safety in algalcultivation were evaluated. Results demonstrated that,(1) the LC100(0.316mg/L) ofcelangulin exterminated the B. plicatilis rotifers effectively in the Nannochloropsis gaditanacultures in24hours. The celangulin treatment had no significant toxicity to the netphotosynthetic and respiratory rates of algal cells. Though partial decreases of chlorophyllcontent were observed, the N. gaditana kept growing during the cultivation period, with theharvest dry biomass reaching84%of the control;(2) Compared with the celangulin,toosendanin showed stronger chronic toxicity on inhibiting the population growth of rotifers.The LC30~LC50(1.755~2.132×10-3mg/L) of toosendanin decreased the rotifer density in48~72h, with the obvious antifeeding responses of survivals in the Chlorella sp. and N.oceanica cultures. Thus, both the population density and egg ratio of survival rotifers kept inlow levels. The toosendanin treatments had no negative influences on the photosyntheticefficiency of algal cells. There was no significant difference on the average specific growthrate between the algae treated by toosendanin and the control. Celangulin and toosendaninshowed none or low toxicity to non-target organisms in aquatic environments. Together withtheir advantages of rapid degradation, low residue and low commercial price, celangulin andtoosendanin are considered to be good potential botanical pesticides for controlling rotifersin microalgal mass cultivation.3. Interaction toxicity of binary combinations between celangulin, matrine andtoosendanin were dependent on the pesticides themselves and their ratios in combinations.Mixtures of matrine/toosendanin mainly produced addition owing to their similar modes ofaction aiming at the nervous system. As the content of matrine or toosendanin incombinations increased, there was an increased trend in the binary toxicity of either celangulin/matrine or celangulin/toosendanin combinations. Combinations of celangulinmixed with matrine or toosendanin at the1:9ratio exhibited synergism, which was attributedto the interference of matrine or toosendanin with the detoxification enzymes of celangulin.The synergistic celangulin/matrine (1:9) or celangulin/toosendanin (1:9) treatment will leadto increased insecticidal efficacy, reduced dosage of biocides, thereby reducing the cost ofexterminating rotifers.4. Based on the management strategy of frequent low-dose treatments, four dailytreatments of0.006mg/L celangulin/toosendanin (CA/TSN)(1:9) combination reduced thepopulation density of rotifers to approximately one rotifers/ml in four days. Thelow-concentration treatments do not directly cause rotifer death, but rather causephysiological disorders by damaging the digestive system, paralyzing the neuromuscularsystem and finally inhibiting energy uptake of the rotifers. Four additions of0.006mg/LCA/TSN treatment had no inhibitory effects on the effective quantum yield (φPSII) andphotosynthetic efficiency (α) of Chlorella sp. and N. oceanica. The microalgae treated by thecombination kept growing, with the pigment content reaching70~90%of the control.Application of the synergistic CA/TSN combination leads to an obvious dosage reduction by30~90%of celangulin and toosendanin for rotifer elimination. As a result, the potentialtoxic impacts for non-target organisms were decreased. The cost for rotifer exterminationwould also be reduced by29%.5. The outdoor experiments showed that two additions of0.003~0.006mg/L of thecelangulin/toosendanin (CA/TSN)(1:9) combination effectively inhibited rotiferreproduction within3days, and showed no toxicity toward the algal biomass, including thechlorophyll and phycocyanin levels of S. platensis. A comparison between the CA/TSNcombination and NH4HCO3treatments indicated that0.003mg/L CA/TSN showed a lastingeffect on controlling rotifers by sterilizing the rotifers. In the NH4HCO3treatment, therotifers still reproduced at a low level, partially owing to the dissipation of thetreatment-generated ammonia. Neither the NH4HCO3nor the CA/TSN treatment changed algal filament morphology; however, the NH4HCO3treatment initially inhibited algal growthmore seriously. S. platensis treated by CA/TSN reached a higher biomass concentration inthe same time frame. Given its effectiveness, together with the projected increase in farmearnings, the synergistic CA/TSN combination showed excellent potential and economicfeasibility for rotifer extermination in outdoor mass cultures of S. platensis.6. The insecticidal mechanism of toosendanin for rotifers was preliminarily studied.Results suggested that LC30~LC70(1.755~2.59×10-3mg/L) of toosendanin seriouslywrinkled the body wall of rotifers by isomerizing the membrane proteins in the epidermalcells, thereby resulting in the imbalance of osmotic pressures inside and outside of cells.Toosendanin decreased the body length and width, and shrunk the body size of survivalrotifers significantly. For the digestive enzymes, toosendanin decreased the rate ofpepsase-catalyzed reaction by improving the binding but inhibiting the further decomposingof pepsase and substrates. In contrast, toosendanin decreased the rate of tryptase-catalyzedreaction by reducing the affinity of tryptase with substrates. The inhibition of toosendanin onpepsase and tryptase activities resulted in the obvious antifeeding responses of rotifers,which was dosage-dependent.