Combined treatment of toxic cyanobacteria Microcystis aeruginosa with hydrogen peroxide and microcystin biodegradation agents results in quick toxin elimination.

Under some conditions the growth of toxic cyanobacteria must be controlled by treatment with algicidal compounds. Hydrogen peroxide has been proposed as an efficient and relatively safe chemical which can remove cyanobacteria from the environment selectively, without affecting other microorganisms. However, the uncontrolled release of secondary metabolites, including toxins may occur after such a treatment. Our proposal presented in this paper concerns fast biodegradation of microcystin released after cell lysis induced by hydrogen peroxide. The effectiveness of both, Sphingomonas sp. and heterologously expressed MlrA enzyme, in the removal of the toxin from Microcystis aeruginosa culture was investigated. The results indicate that neither Sphingomonas cells nor MlrA are affected by hydrogen peroxide at the concentrations which stop the growth of cyanobacteria. A several-fold reduction in microcystin levels was documented in the presence of these agents with biodegradation ability. Our results provide evidence that such a combined treatment of water reservoirs dominated by microcystin-producing cyanobacteria may be a promising alternative which allows fast elimination of both, the bloom forming species and toxins, from the environment.

Heterologous expression of mlrA in a photoautotrophic host - Engineering cyanobacteria to degrade microcystins.

In this report, we establish proof-of-principle demonstrating for the first time genetic engineering of a photoautotrophic microorganism for bioremediation of naturally occurring cyanotoxins. In model cyanobacterium Synechocystis sp. PCC 6803 we have heterologously expressed Sphingopyxis sp. USTB-05 microcystinase (MlrA) bearing a 23 amino acid N-terminus secretion peptide from native Synechocystis sp. PCC 6803 PilA (sll1694). The resultant whole cell biocatalyst displayed about 3 times higher activity against microcystin-LR compared to a native MlrA host (Sphingomonas sp. ACM 3962), normalized for optical density. In addition, MlrA activity was found to be almost entirely located in the cyanobacterial cytosolic fraction, despite the presence of the secretion tag, with crude cellular extracts showing MlrA activity comparable to extracts from MlrA expressing E. coli. Furthermore, despite approximately 9.4-fold higher initial MlrA activity of a whole cell E. coli biocatalyst, utilization of a photoautotrophic chassis resulted in prolonged stability of MlrA activity when cultured under semi-natural conditions (using lake water), with the heterologous MlrA biocatalytic activity of the E. coli culture disappearing after 4 days, while the cyanobacterial host displayed activity (3% of initial activity) after 9 days. In addition, the cyanobacterial cell density was maintained over the duration of this experiment while the cell density of the E. coli culture rapidly declined. Lastly, failure to establish a stable cyanobacterial isolate expressing native MlrA (without the N-terminus tag) via the strong cpcB560 promoter draws attention to the use of peptide tags to positively modulate expression of potentially toxic proteins.

The biodegradation of microcystins in temperate freshwater bodies with previous cyanobacterial history.

Cyanobacterial blooms and cyanotoxins occur in freshwater lakes and reservoirs all over the world. Bacterial degradation of microcystins (MC), hepatotoxins produced by several cyanobacterial species, has also been broadly documented. However, information regarding MC biodegradation in European water bodies is very limited. In this paper, the occurrence and identification of MC biodegradation products was documented for 21 European lakes and reservoirs, many of which have well-documented cyanobacterial bloom histories. Varying cyanobacterial abundance and taxonomical composition were documented and MC producers were found in all the analysed samples. Planktothrix agardhii was the most common cyanobacterial species and it formed mass occurrences in four lakes. MC biodegradation was observed in 86% of the samples (18 out of 21), and four products of dmMC-LR decomposition were detected by HPLC and LC-MS methods. The two main products were cyclic dmMC-LR with modifications in the Arg-Asp-Leu region; additionally one product was recognized as the tetrapeptide Adda-Glu-Mdha-Ala. The composition of the detected products suggested a new biochemical pathway of MC degradation. The results confirmed the hypothesis that microcystin biodegradation is a common phenomenon in central European waters and that it may occur by a mechanism which is different from the one previously reported. Such a finding implies the necessity to develop a more accurate methodology for screening bacteria with MC biodegradation ability. Furthermore, it warrants new basic and applied studies on the characterization and utilization of new MC-degrading strains and biodegradation pathways.