Introduction: Harmful algal blooms (HABs) commonly cause mass mortalities of fish, shellfish, and other freshwater life due to toxin production and depletion of oxygen. Repeated and widespread HABs can drastically change freshwater ecologies. Furthermore, ingestion of freshwater-foods with algal toxins can lead to illness and death in humans and animals, and exposure to aerosolized toxins from contaminated water causes respiratory problems in humans[1]. For these reasons, it is important to develop materials and methods to prevent or control HABs in freshwater environments, aquaculture industry, and human health.
Chitosan was recently used to convert sand into effective flocculants for mitigation of HABs in marine and freshwater systems and soil/clay flocculation. However, there have been no reports of the use of chitosan alone for removing and controlling HAB[2]. We investigated the selective algicidal effect of Chitosan with various molecular weight against green-tide. Furthermore, the mode of algicidal action, molecular weight range, selective killing for harmful green-tide, non-cytotoxic effects, and recovered survival of freshwater in the presence of an artificial green-tide were demonstrated.
Experimental Methods: Chitosan solution was prepared by using lactic acid as a solvent. 5 units of chitosanase originated from Bacilllus pumilus BN-262 was added to 100 mL of the said 5% chitosan solution (pH 5.0 ~ 5.5), followed by enzyme reaction at 40 ℃ for 36 hours. Upon completion of the reaction, the mixture was filtered by using a 1 μm pre-filter and then filtered again by using a hollow filter. The obtained filtrate was concentrated by using the nanofilter system, followed by sterilization and drying with an air spray dryer. As a result, chitosan polysaccharide was obtained.
The cytotoxicity of chitosan was evaluated using an MTT assay in L929 cell lines. In addition, anti-green algae activity using various molecular weight chitosan was confrimed by inhibitory concentration (IC50).
Results and Discussion: Cytotoxicity of chitosan was confirmed by MTT assay. The results showed that cell viability of high molecular chitosan (780kDa) and low molecular chitosan (10kDa) was measured above 70% and mentioned harmless materials.
To confirm algicidal activity of chitosan with various molecular weight, we investigated by inhibitory concentration (IC50). The results showed that algicidal activity of chitosan (Mw:780 kDa) against microcystis, anabaena, and chlorella was indicated such as 0.012g/L, 0.024 g/L and 0.012 g/L, respectively. In addition, algicidal activity in case of chitosan (Mw: 10 kDa) was indicated such as 0.39 g/L, 0.097 g/L, and 0.049 g/L, respectively. These results mean that algicidal effect of high molecular weight chitosan was better than low molecular chitosan. For these reason, chitosan free-amine of positive charge was affected to membrane with negative charge of green-tide. Moreover, free-amine of high molecular weight chitosan was existed more than low molecular weight chitosan. Therefore, chitosan has a potential ability of control without cytotoxicity at green-tide.
Conclusion: In this study, we prepared chitosan for removed green-tide. The cytotoxicity of prepared chitosan was not indicated. In addition, algicidal activity of chitosan was confirmed by inhibitory concentration (IC50). Theses results suggest that chitosan can be utilize as material of protecting freshwater ecology from green-tide.
This work was supported by National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT & Future Planning. (No. NRF-2014R1A2A1A10053027).
References:
[1] D.M. Anderson et al., Annual review of marine science, 4(2012), 143-176.
[2] Li, L et al., Environmental Science & Technology, 47(2013), 4555-4562.