Final Report for the Lake Erie Protection Fund Small Grants Program Identification of Microcystin-Degrading Bacteria (SG 374-09)

The main objective of this project is to investigate the role of heterotrophic bacteria in degrading one of the major cyanotoxins, e.g., microcystin, that are produced during harmful cyanobacterial algal bloom events (cyanoHABs) in the western basin of Lake Erie. Microcosm incubation experiments were set up to study response of lake bacterioplankton (cells that pass 1.0 μm-pore size filters) to elevated supply of microcystin LR (10 μg/L, final concentration). After 48 hr incubation, bacterial abundance in the microcystin amended microcosms (MCs) increased nearly 10 folds, while remained unchanged in the no-addition controls (CTRLs). At the same time, the microcystin concentration in the MCs decreased 70%, indicating active consumption of microcystin by bacterioplankton in the MCs. Genomic materials of bacterioplankton communities in the MCs and CTRLs were sequenced. Bioinformatic analysis identified a set of gene categories that were significantly enriched in the MCs relative to the CTRLs. Analysis of significantly enriched gene sequences indicated that microcystin degradation in Lake Erie may involve a diverse group of heterotrophic bacterioplankton, with Betaproteobacteria of particular importance. This was different from previous studies in other freshwater systems, which indicated bacteria of the Actinobacteria and Alphaproteobacteria taxa were most important in microcystin degradation. Lake Erie Protection Fund SG374-09 “Identification of Microcystin-Degrading Bacteria” Final Technical Report • May 2011 Page 2 of 9 INTRODUCTION Lake Erie supplies drinking waters to millions of human and is home to numerous wild lives. Therefore, maintaining and improving the quality of Lake Erie water is of the top priority of local, state and federal management agencies. However, despite much research and restoration efforts, harmful cyanobacterial algal blooms (cyanoHABs) recur annually in Lake Erie, especially in the Western Basin, with increasing frequency, extent and affected areas. CyanoHABs produce a number of substances that are toxic to humans and lake organisms. One class of these cyanotoxins, microcystins, is particularly pernicious due to its damaging effects on animal liver. In warm months (June, July and August), microcystin concentrations in the Lake Erie Western Basin frequently exceed the World Health Organization (WHO) guideline values of drinking (1 μg/L) and recreational bath waters (low risk at 2-4 μg/L) (Figure 1), which is one of the major factors causing beach closing. The toxicity of microcystins is determined by their concentrations in the water, so processes that produce and remove these toxins are equally important in controlling the hazardous effects of cyanoHABs. Early studies on the microcystin production rely on tracking their concentration inside of cyanobacterial cells under varied growth conditions (Chorus, 2001). Recent identification of the key genes involved in microcystin biosynthesis has promoted such studies to the genetic level (Kaebernick et al., 2000; Kaebernick et al., 2002). It is clear now that Microcystis cells regulate the production of microcystin under conditions of different cell growth phase, light quality and intensity, temperature and nutrient supplies (nitrogen, phosphorous and iron) (Ouellette and Wilhelm, 2003). What is still not clear is the opposite process, the degradation and removal of microcystins after they released into lake waters. This knowledge gap prohibits precise prediction of the overall harmfulness of cyanobacteria bloom events, which is necessary for efficient management efforts. Moreover, this knowledge gap limits our understanding on the mechanism of the ecosystem to naturally recover from a distorted food web, which has great implication to practical and environmental friendly bioremediation. Studies have shown that abiotic reactions like photolysis are only responsible for part of the removal of microcystin, and these reactions usually take days or months under natural NOAA Western Lake Erie Water sampling Stations Figure 1. Microcystin concentrations in Lake Erie Western Basin frequently exceed the safe use levels in summer 2010 (data from NOAA, Harmful Algal Bloom Events Response; http://www.glerl.noaa.gov/res/Centers/HABS/western_lake_erie.html). This study Lake Erie Protection Fund SG374-09 “Identification of Microcystin-Degrading Bacteria” Final Technical Report • May 2011 Page 3 of 9 conditions (Welker and Steinberg, 2000). Heterotrophic bacteria are known for their strong ability to decompose a variety of naturally produced or synthetic organic compounds that are commonly found within their native habitat. The positive role of bacterial community in degrading microcystin has been indicated by several previous studies in other water bodies (Christoffersen et al., 2002; Ho et al., 2007; Lemes et al., 2008). However, clear linkage between bacterial taxa and in situ microcystin degradation is yet to be discovered. The lack of knowledge on genes and pathways that involved in the processes hinders our ability in sensitive and specific monitoring microcystin degradation in environmental samples. The goal of this project is to solve these problems using an experimental metagenomic approach. Our results suggest that a diverse group of lake bacterioplankton are involved in microcystin degradation, with Betaproteobacteria play particularly important roles.