Microcystin Pollution
Cyanobacteria are found everywhere: they thrive in lakes, reservoirs, rivers, oceans, and sometimes on land. Cyanotoxins are secondary metabolites of the cyanobacteria. Among them, microcystin is one of the most toxic: several case studies have proved its potential to cause acute liver failure and liver cancer, and microcystin-LR (the most toxic among over 250 types) has been classified as a group 2B carcinogen. Upon ingestion through oral intake, inhalation or injection, they travel to the liver via the bile transport system and demonstrate hepatoxicity by inhibiting the function of protein phosphatase PP1 and PP2A.
Large quantities of microcystins can be produced during harmful algal blooms, threatening water quality. The contaminated water may enter the food chain via irrigation or directly harm people who drank it. In 1996, Microcystin-LR present in the water for hemodialysis treatment led to the death of over 60 patients in Caruaru, Brazil. (Jochimsen et al., 1998) This incident greatly helped gather attention on microcystin pollution issues and eventually led World Health Organization to include microcystin-LR in its Guidelines to Drinking Water Quality, with 1 µg/L as the provisional guide value.
Traditional Degradation Methods
Microcystins are cyclic heptapeptides. This structure makes them resistant to changes in temperature or pH, microwave radiation, and non-specific enzymes. They therefore require more advanced approaches.
Coagulation and Filtration are representative of the conventional water treatments. They can remove cyanobacteria cells, but fail to directly address the toxins released in the environment.
Adsorption with either powdered or granular activated carbon effectively remove microcystins, yet it is incapable of transforming the toxin into harmless by-products, so the challenge to treat the retained toxins remain. Consumable parts in the system require frequent changing. The adsorbents are also susceptible to other organic molecules present in the water.
Chemical Oxidation and Advanced Oxidation Processes effectively decompose the structure of microcystins through the addition of oxidants or photocatalysis. (Liang et al., 2021) However, both methods yield harmful or unidentified intermediate products that could cause serious secondary pollution. The reagents involved in chemical oxidation are pollutive as well. Advanced oxidation processes require significant functional costs, complex technological requirements and high maintenance, all of which make it unsuitable for implementation in suburban or rural areas where harmful algal blooms are most potent. (Dong et al., 2012)
The relatively new field of bacterial degradation could prove helpful. It meets the 3 most crucial criteria for larger scale microcystin degradation: 1) high removal efficiency; 2) less technical operation; 3) low cost. They efficiently degrade dissolved microcystins without generating pollutive metabolites, unlike other physical or chemical treatments. Being environmentally-friendly in nature, they not only solve the imminent issue of high microcystin concentration, but may also address the ecological imbalance caused by harmful algal blooms and prevent future microcystin pollution. (Ding et al., 2022)
Natural Biodegradation
The first identification of microcystin degrading bacteria in 1994 marked the beginning of the genetic approach to the toxin. Since then, dozens of novel bacteria have been identified and isolated from water bodies. Most of them possessed the mlr gene cluster, which had been sequenced and verified to be responsible for the natural microcystin degradation pathway. MlrABC are coding sequences for enzymes in the cascade, while mlrD is an oligopeptide that uptakes microcystins into the bacteria cell.
The studies of the pathway, although universal to all microcystins, usually take microcystin-LR, the most toxic variety, as example. “LR” referred to the 2 distinct amino acids in the heptapeptide.
The first step in the pathway is linearization of the cyclic microcystin. MlrA catalyzes the process and cleaves the peptide bond between Adda and L-Arg. MlrB then proceeds to cleave the peptide bond between D-Ala and L-Leu, creating a tetrapeptide and a tripeptide. MlrC finally separates the amino acid Adda, which is responsible for the toxicity in microcystins.
Our design
The 1st step in the pathway, linearization, is the most efficient one. Although values reported by studies vary, it can be ascertained that the by-product, linearized microcystin, is significantly lower in toxicity than the cyclic microcystin, and can be considered non-toxic for environmentally relevant concentrations. (Dziga et al., 2012)
We decide to take advantage of this reaction and explore a simpler degradation system. Through review of literature, we found that the ice nucleation protein (inaK) is often used to display enzymes at the surface of recombinant bacteria, enabling it to directly degrade chemicals in the environment. (Liu et al., 2020) We believe that our MlrA enzyme would be compatible with the cross-membrane InaK protein, and the extracellular enzyme display system worked to our interest in directly removing dissolved microcystins in the environment.
References:
[1] Jochimsen, E., Carmichael, W., An, J., Cardo, D., Cookson, S., Holmes, C., Antunes, M., de Melo Filho, D., Lyra, T., Barreto, V., Azevedo, S. and Jarvis, W., 1998. Liver Failure and Death after Exposure to Microcystins at a Hemodialysis Center in Brazil. New England Journal of Medicine, 338(13), pp.873-878.
[2] Xiang, L., Li, Y., Liu, B., Zhao, H., Li, H., Cai, Q., Mo, C., Wong, M. and Li, Q., 2019. High ecological and human health risks from microcystins in vegetable fields in southern China. Environment International, 133, p.105142.
[3] Ding, Q., Song, X., Yuan, M., Sun, R., Zhang, J., Yin, L. and Pu, Y., 2022. Removal of microcystins from water and primary treatment technologies – A comprehensive understanding based on bibliometric and content analysis, 1991–2020. Journal of Environmental Management, 305, p.114349.
[4] Li, J., Li, R. and Li, J., 2017. Current research scenario for microcystins biodegradation – A review on fundamental knowledge, application prospects and challenges. Science of The Total Environment, 595, pp.615-632.
[5] Dong, W., Sun, Y., Ma, Q., Zhu, L., Hua, W., Lu, X., Zhuang, G., Zhang, S., Guo, Z. and Zhao, D., 2012. Excellent photocatalytic degradation activities of ordered mesoporous anatase TiO2–SiO2 nanocomposites to various organic contaminants. Journal of hazardous materials, 229, pp.307-320.
[6] Liang, D., Li, N., An, J., Ma, J., Wu, Y. and Liu, H., 2021. Fenton-based technologies as efficient advanced oxidation processes for microcystin-LR degradation. Science of the Total Environment, 753, p.141809.
[7] Shimizu, K., Maseda, H., Okano, K., Kurashima, T., Kawauchi, Y., Xue, Q., Utsumi, M., Zhang, Z. and Sugiura, N., 2012. Enzymatic pathway for biodegrading microcystin LR in Sphingopyxis sp. C-1. Journal of Bioscience and Bioengineering, 114(6), pp.630-634.
[8] Dziga, D., Wladyka, B., Zielińska, G., Meriluoto, J. and Wasylewski, M., 2012. Heterologous expression and characterisation of microcystinase. Toxicon, 59(5), pp.578-586.
[9] Liu, M., Feng, P., Kakade, A., Yang, L., Chen, G., Yan, X., Ni, H., Liu, P., Kulshreshtha, S., Abomohra, A.E.F. and Li, X., 2020. Reducing residual antibiotic levels in animal feces using intestinal Escherichia coli with surface-displayed erythromycin esterase. Journal of hazardous materials, 388, p.122032.