| In recent years, with the rapid development of coastal area economy and aquaculture industry, water environment pollution is increasingly serious. Red tide/harmful algal blooms (HABs) have been occured frequently in various coastal area, lakes and reservoirs, and the affected regions increased gradually. Therefore, it is extremely urgent to explore the effective, economical and environment-friendly eutrophication control methods. For controlling cyanobacteria over-growing, biological control methods have been recently explored. Antialgal microbial technology has been regarded as a novel and safe method for eutrophication remediation because of its low cost, low secondary pollution risk characteristics and high efficiency. Based on the advantages of antialgal microbial technology, the antialgal bacteria with highly effective inhibiting activity were isolated from eutrophication waters in Zhejiang province using Microcystis aeruginosa as a model species. The molecular identification, physiological and biochemical characteristics, the optimization of the culture medium and fermentation conditions of antialgal bacterium strain HJC-D1, the characteristics and acute toxicity of antialgal products, antialgal process and mechanism, and algae and nitrogen removal performance of biofilm process with the addition of strain HJC-D1from micro-polluted source water were investigated in this paper, the main results are as follows:1. Four antialgal bacteria (HJC-D1, ZJG-24, ZJG-215and KY-63) which exhibited antialgal activity against a representative bloom-forming cyanobacterium M. aeruginosa were isolated from eutrophication waters, and the antialgal effect of strain HJC-D1was the best. Based on its morphology, physiological and biochemical characteristics and16S rRNA sequence, the isolated strain was identified as Streptomyces sp. The best antialgal effect was obtained as the strain HJC-D1fermented for4d, and the M. aeruginosa could be inhibited by the isolated strain and the Chi a removal efficiency was53.5±4.3%～62.6±3.3%at the initial Chi a concentration of0.2713～0.6375mg L-1.The antialgal active substances mainly existed in extracellular aseptic filtrate, with thermal stability, basophilic and could be extracted by ethyl acetate. In such a way it was speculated that the antialgal way of Streptomyces sp. HJC-D1was indirect effect. Based on the above research results, in order to improve the antialgal effect and raise the production of antialgal active substances, a sequential optimization of the culture medium based on statistical design was employed. Sucrose and KNO3were selected as the most suitable carbon and nitrogen sources based on the one-factor-at-a-time method. Sucrose, KNO3and initial pH were found to be the major impact factors for further optimization obtained from the Plackett-Burman design. The optimum levels for these key variables were analysed by central composite design, and the optimum condition of the culture medium was obtained as following (g·L-1):sucrose,22.73; KNO3,0.96; NaCl,0.5; K2HPO4,0.5, MgSO4,0.5; FeSO4,0.002, and with initial pH of8.8; On the basis of the optimised culture medium, the influence of fermentation conditions such as temperature, initial pH, volume and agitation rate were carried out, and the best level of operating parameters was33.1℃for temperature,11.8for initial pH,91.2mL for volume and337.5rpm for agitation rate. Additionly, the maximizing removal efficiency of M. aeruginosa under the optimized conditions in flask cultures was93.7%.2. Using fourier transform infrared spectroscopy (FTIR),3-D fluorescence spectroscopy (EEMs) and gel permeation chromatography (GPC), the composition and characteristics of dissolved organic matter (DOM) from antialgal products were studied. The results indicated that the growth of M. aeruginosa could be effectively inhibited as the addition scale of HJC-D1fermentation cultrue was above3%(v/v) at a initial Chl a concentration of0.2835±0.0721mg·L-1, and the removal efficiency was72.6±5.5%and82.8±1.8%with the addition dosage of5%and10%, respectively. The main fluorescent substance in DOM was humic-like acid, and the molecular weight of antialgal products was around1000Da. The antialgal products were divided using resin adsorbents into hydrophilic fraction (HPI), hydrophobic acid (HPO-A), transphilic acid (TPI-A), hydrophobic neutral and transphilic neutral, and the results indicated that HPI component was the most abundant DOM fraction in the antialgal products, and the HPI concentration was increased with the increasing of fermentation liquid concentration. The fluorescence peak location and intensity showed that the protein-, fulvic-and humic-like substances were dominant in HPI, HPO-A and TPI-A fractions, and intensities of the relevant fluorescence peaks were stronger in experimental groups than those in control groups; it was also found that the number averaged molecular weight of DOM fractions ranged from245to1452g mol-1, and thereinto organic acids such as HPO-A and TPI-A exhibited lower molecular weights, and the adsorption band at1031cm-1for TPI-A corresponded to carbohydrates and polysaccharide-like substances. The acute toxicity of Streptomyces sp. HJC-D1fermentation broth and antialgal products was studied with Photobacterium phosphoreum, the results indicated that the fermentation broth of antialgal bacterium had certain biotoxicity via photobacteria tests, and the relative luminosity was67.6±3.1%～72.4±2.8%after3-5d fermentation. The toxicity tests with Microcystin-LR as the main antialgal product showed that there’s no inhibited effect when the Microcystin-LR concentration was lower than20μg·L-1.3. The cell integrity and physiological characteristics of M. aeruginosa, antioxidant enzymes and oxidative damage change were investigated to illustrate the antialgal mechanism of M. aeruginosa by strain Streptomyces sp. HJC-D1. Results showed that the growth of M. aeruginosa was significantly inhibited, and the percentage of live cells depended on the fermentation liquid concentration and exposure time. The percentage of live algal cells in the3%,5%and10%treatment groups were51.4%,15.7%and2.3%after8d incubation, respectively. Furthermore, TEM observation suggested the cell wall of M. aeruginosa was damaged dramatically during the antialgal process, and the permeability of cell membrane was increased with the release of K+and Ca2+; Under the stress of aseptic filtrate of Streptomyces sp. HJC-D1, the activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were significantly changed, which was increased with the increasing of exposure concentration and the extending of exposure time, and the significant increase in the lipid-peroxidation product malondialdehyde (MDA) triggered the synthesis of reactive oxygen species (ROS) to disrupt the subcellular structure of M. aeruginosa, and organelles were released which resulted in the death of algae cell finally. Streptomyces sp. HJC-D1therefore not only affected antioxidant enzyme activities and ROS level, but also destroyed the subcellular structure of M. aeruginosa. According to the above results, it was speculated that the antialgal mechanism was as following:the algal cell wall was destroyed by antialgal active substances that secreted by antialgal bacterium, then cell organelle such as thylakoid, photosynthetic lamellar and phosphate particles were released that leading to inactivation, and resulted in the death of algae cells finally.4. Facing increasingly serious problem of micro-polluted water by harmful algae blooms in our country, strain Streptomyces sp. HJC-D1, which was capable of inhibiting M. aeruginosa, was immobilized to AquaMats to evaluate the removal performance of algae and denitrification by biofilm reactor. The results showed that the average removal efficiency of chlorophyll α, NH4+-N and CODMn was71.7±5.4%,77.8±2.8%and25.0±1.5%for the control reactor R1, and80.9±4.4%,72.4±3.2%and18.7±1.4%for the experimental reactor R2, respectively. On the condition of aerobic to anoxic ratio of2h:4h and aeration intensity of60L-h"1, the removal efficiencies of CODMn and NH4+-N were improved in the control group, while the Chi a removal efficiency was not changed significantly, and the accumulation of NO3--N for the control group was much more significantly; The change of the hydraulic retention time (HRT) had little impact on the removal performance of pollutants such as NH4+-N and CODMn, while the Chi a removal efficiency was decreased with HRT; Microscopic observation revealed that the surface of AquaMats in experimental reactor was surrounded with antialgal bacterium. It was presumed that the removal mechanism of algae for the control group mainly through the adsorption of AquaMats, while the algae was absorbed by AquaMats and then degraded by antialgal strain Streptomyces sp. HJC-D1for the experimental reactor.In conclusion, according to the studies of the antialgal characteristics, the antialgal mode of Streptomyces sp. HJC-D1, the characteristics of antialgal degradation products and the enhanced performance of immobilized microorganism technology for algae remonal and denitrification effect with micro-polluted surface watere, it was noteworthy that antialgal substances produced by Streptomyces sp. HJC-D1had the potential to be developed as a commercial biological control agent and it might provide a feasible bioremediation technology for the control of algal blooms in our country. |
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