Article pubs.acs.org/est
Kinetics of Cell Inactivation, Toxin Release, and Degradation during Permanganation of Microcystis aeruginosa Lei Li,† Chen Shao,† Tsair-Fuh Lin,‡ Jiayu Shen,† Shuili Yu,† Ran Shang,§ Daqiang Yin,∥ Kejia Zhang,⊥ and Naiyun Gao*,† †
State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai City 200092, China Department of Environmental Engineering, National Cheng Kung University, Tainan 70101, Taiwan § Department of Sanitary Engineering, Faculty of Civil Engineering and Geoscience, Delft University of Technology, Post Office Box 5048, 2600 GA Delft, The Netherlands ∥ Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Engineering, Tongji University, Shanghai 200092, China ⊥ Department of Civil Engineering, Zhejiang University (Zijingang Campus), Hangzhou 310058, China ‡
S Supporting Information *
ABSTRACT: Potassium permanganate (KMnO4) preoxidation is capable of enhancing cyanobacteria cell removal. However, the impacts of KMnO4 on cell viability and potential toxin release have not been comprehensively characterized. In this study, the impacts of KMnO4 on Microcystis aeruginosa inactivation and on the release and degradation of intracellular microcystin-LR (MC-LR) and other featured organic matter were investigated. KMnO4 oxidation of M. aeruginosa exhibited some kinetic patterns that were different from standard chemical reactions. Results indicated that cell viability loss and MC-LR release both followed two-segment second-order kinetics with turning points of KMnO4 exposure (ct) at cty and ctr, respectively. KMnO4 primarily reacted with dissolved and cell-bound extracellular organic matter (mucilage) and resulted in a minor loss of cell viability and MC-LR release before the ct value reached cty. Thereafter, KMnO4 approached the inner layer of the cell wall and resulted in a rapid decrease of cell viability. Further increase of ct to ctr led to cell lysis and massive release of intracellular MC-LR. The MC-LR release rate was generally much slower than its degradation rate during permanganation. However, MC-LR continued to be released even after total depletion of KMnO4, which led to a great increase in MC-LR concentration in the treated water.
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INTRODUCTION In the past decades, the detection of cyanobacterial (or bluegreen algae) blooms has been more common worldwide.1 Cyanobacteria and the generation of algogenic organic matter (AOM), including intracellular organic matter (IOM) and extracellular organic matter (EOM), are continuously critical challenges in drinking water treatment. A variety of preoxidation techniques including chlorination, ozonation, potassium permanganation (KMnO4), and UV irradiation are usually applied to maximize the removal of cyanobacteria cells. However, these technologies have resulted in the release of problematic byproducts, including intracellular toxins such as microcystin-LR (MC-LR), disagreeable taste and odor (T/O) compounds, and other types of AOMs.2−4 These adverse effects have caused a great number of water treatment operational problems.2,5−7 There have been several studies on cyanobacteria preoxidation and the subsequent cell damage and IOM release.1,8−11 © 2014 American Chemical Society
However, previous work has mainly focused on prechlorination. It is reported that chlorine could easily cause serious cell lysis and IOM release even at very low chlorine dosages.1,9,10 Since cell wall damage (k = 670 ± 77 M−1·s−1) occurs faster than the degradation of released algae toxin (k = 242 M−1·s−1),9 extracellular toxin or T/O compounds would accumulate to dangerously high concentrations after chlorination.9,10,12 In addition, chlorine preoxidation has been reported to produce regulated disinfection byproducts (DBPs).1 In comparison to chlorine oxidation, cyanobacteria cells are less sensitive to KMnO4 oxidation,13 and KMnO4 oxidation likely results in less cell damage and IOM release.14 Moreover, the released IOM tends to be easier to remove with KMnO4 through chemical Received: Revised: Accepted: Published: 2885
November 27, 2013 February 2, 2014 February 6, 2014 February 6, 2014 dx.doi.org/10.1021/es405014g | Environ. Sci. Technol. 2014, 48, 2885−2892
Environmental Science & Technology
Article
Preoxidation Experiments and Models. During the oxidation of M. aeruginosa by KMnO4, two indicative parameters, MC-LR and cell viability, were tested. Aqueous MC-LR concentration (i.e., extracellular MC-LR) is affected by at least three factors: direct oxidation by KMnO4,26 adsorption by newly formed MnO2, and the release of intracellular MCLR.14 The reaction constants of the above three processes were separately studied: (1) MC-LR degradation constant was studied by mixing MCLR-spiked EOM and IOM with KMnO4 [initial concentrations of MC-LR, KMnO4, and dissolved organic carbon (DOC) in the EOM or IOM were 200 μg/L, 5 mg/L, and 5 mg/L respectively]. Samples were taken at intervals to quantify the remaining MC-LR and KMnO4. (2) The adsorption behavior of MC-LR by MnO2 was studied by mixing MC-LR-spiked IOM and EOM solution with newly made MnO2 (MnO2 was obtained through the reaction of 40 mg/L KMnO4 with Na2S2O3 at pH 7.5. Other chemical conditions were the same as those for process 1). Then, samples were taken at intervals to quantify the remaining MCLR. (3) To determine the release constant of intracellular MCLR, the harvested M. aeruginosa was treated with KMnO4. Residual KMnO4 and intracellular MC-LR were quantified at intervals. The overall aqueous MC-LR was then calculated as
oxidation as well as adsorption/coprecipitation with the newly formed manganese dioxide (MnO2).15,16 However, little work has been done to systematically study the kinetics of these KMnO4 reactions. One reason that knowledge of cyanobacteria preoxidation is so limited is the lack of effective technology sets to comprehensively address the inactivation and removal of cyanobacteria cells and the release of IOM during oxidation.14 Several methods, such as flow cytometry,9,17,18 scanning and transmission electron microscopy (SEM and TEM),18,19 and pulse amplitude modulation (PAM) fluorometry,20,21 have been used to assess cyanobacteria cell viability and cell wall damage/integrity. With flow cytometry, stains were applied to react with the algae nucleic acid, causing the damaged or nonviable cells to fluoresce prior to measurement of fluorescence and statistical analysis. Other studies have used SEM/TEM to qualitatively document the physical destruction of various cyanobacteria after oxidation. However, SEM/TEM detection requires several complicated steps of preparation and captures only a few cells per image. Recently, PAM fluorometry was reported to provide an excellent representation of the cell activity of cyanobacteria by monitoring the changes of fluorescence parameters (e.g., effective quantum yield, Φ).20 Our previous studies demonstrated that Φ provided a good explanation of the immediate and long-term impacts of oxidant on photosynthetic viability of Microcystis aeruginosa.21,22 Additionally, an overview of AOM transformation, particularly IOM release, is important when estimating the adverse effects of preoxidation technology. Fluorescence excitation−emission matrix (EEM) is a rapid, selective, and sensitive technique used to distinguish the organic compounds in water.23 Most AOM of cyanobacteria, including proteins, enzymes, coenzymes, pigments, and metabolites, have fluorescence properties and exhibit fluorescent emission−excitation combinations in specific regions. Several studies have documented that EEM provided a complete representation of the fluorescence spectral features of algae samples and enabled a better identification of different AOM fractions.19,24 However, the transformation kinetics of the EEMs of dissolved organic matters during permanganation of cyanobacteria have not been previously reported. In this study, one of the most abundant blue-green algae species, M. aeruginosa, was chosen as the surrogate cyanobacterium.25 Kinetics of cell viability loss and the release of intercellular toxin MC-LR and other featured AOMs during permanganation were comprehensively studied aiming to understand the mechanism of algae inactivation, and therefore to optimize the treatability of algae-laden water in practical applications. The qualitative and quantitative methodology developed in this research could be easily applied to preoxidation of other single or mixed strains of cyanobacteria with the same or other oxidants.
[MC‐LR e]f = [MC‐LR e]0 − [MC‐LR e]d − [MC‐LR e]a + [MC‐LR i]r
(1)
where [MC-LRe]0, [MC-LRe]d, [MC-LRe]a, and [MC-LRe]f were the concentrations of initial, degraded, adsorbed, and final extracellular MC-LR, respectively, and [MC-LRi]r was the concentration of MC-LR released from the algae cell. Meanwhile, cell viability (indicated by reactive quantum yield, Φ) was immediately determined after each sampling to analyze the kinetics of cell inactivation. EEM properties of EOM at different levels of KMnO4 exposure were analyzed to characterize the overall IOM release. All experimental solutions above were adjusted to a pH of 7.5 ± 0.2 by bubbling with CO2 prior to starting the experiment. The solutions were slowly mixed during reactions. All samples were filtrated by prerinsed 0.7 μm glass filter membrane (Whatman) to remove the suspensions prior to testing for remaining MC-LR, permanganate, and EEM. Fluorescence Spectroscopy. Three-dimensional spectra of EOM after different levels of exposure to KMnO4 were obtained by repeatedly measuring the emission (em) spectra in the range from 250 to 550 nm, and at excitation wavelengths (ex) from 200 to 500 nm, at 5 nm intervals in the excitation domain by use of a fluorescence spectrophotometer (model F4500, Hitachi, Japan). Data were analyzed with Matlab (MathWorks Inc., Natick, MA). Cyanobacteria Cell Viability. Cell viability of M. aeruginosa (indicated by quantum yield, Φ) was immediately analyzed with a Phyto-Pam phytoplankton analyzer (Walz, Germany) subsequent to harvesting from the culture. Chemical Analysis. Microcystis aeruginosa samples were taken and Na2S2O3 was added in excess to quench the residual KMnO4 for MC-LR analysis. Half the quenched sample was immediately filtered through a glass membrane to remove cells for extracellular MC-LR detection. The other half was subjected to freeze/thawing treatment for total MC-LR determination9
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MATERIALS AND METHODS Cyanobacteria Cultivation and AOM Extraction. Microcystis aeruginosa, purchased from the Institute of Hydrobiology, Chinese Academy of Sciences, was cultured in BG11 medium in an incubator at 25 °C. The cell solution with a cell density of ∼6.4 × 106/mL was harvested during its exponential growth period prior to preoxidation experiments. EOM and IOM extraction methods were reported elsewhere.2 Concentrations of MC-LR in raw EOM and IOM extracts were 29 ± 8 and 264 ± 14 μg/L, respectively. 2886
dx.doi.org/10.1021/es405014g | Environ. Sci. Technol. 2014, 48, 2885−2892
Environmental Science & Technology
Article
(i.e., the total of intracellular and extracellular MC-LR). Intracellular MC-LR was calculated by subtracting extracellular MC-LR from total MC-LR. MC-LR was determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS; Thermo TSQ Quantum Access Max) with a basic-C18 HPLC column (100 mm × 2.1 mm, 5 μm, Thermo) according to the method described by Zhou et al.22 Initial and residual KMnO4 in the algae solution at each sampling time was detected by measuring the absorbance at 510 nm in a 10 mm cuvette in a UV/vis spectrophotometer (Hach DR2800) subsequent to filtration by 0.7 μm glass filter membrane (Whatman) to remove the manganese dioxide and other suspended particles. Replicability. All tests were conducted at least in duplicate. The relative standard deviations (RSD) for different batches were normally
