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Simultaneous Microcystis–algicidal and microcystindegrading capability by a single Acinetobacter bacterial strain Hong Li, Hainan Ai, Li Kang, Xingfu Sun, and Qiang He Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03986 • Publication Date (Web): 06 Oct 2016 Downloaded from http://pubs.acs.org on October 10, 2016
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Simultaneous Microcystis–algicidal and microcystin-degrading capability by a single
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Acinetobacter bacterial strain
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Hong Li 1, Hainan Ai 1, Li Kang 1, Xingfu Sun 2, Qiang He 1, *
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Education, Chongqing University, Chongqing 400044, China
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Key Laboratory of Eco-Environment of Three Gorges Region of Ministry of
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Xiamen Municipal Engineering Design Institute CO., LTD, Chongqing 401122, China
*Corresponding Author:
[email protected] Tel: +86 23 65120980; Fax: +86 23 65120980
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ABSTRACT: Measures for removal of toxic harmful algal blooms often cause lysis
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of algal cells and release of microcystins (MCs). In this study, Acinetobacter sp.
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CMDB-2 that exhibiting distinct algal lysing activity and MCs degradation capability
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was isolated. The physiological response and morphological characteristics of
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toxin-producing Microcystis aeruginosa, the dynamics of intra- and extracellular
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MC-LR concentration were studied in an algal/bacterial co-cultured system. The
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results demonstrated that Acinetobacter sp. CMDB-2 caused thorough decomposition
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of algal cells and impairment of photosynthesis within 24 h. Enhanced algal lysis and
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MC-LR release appeared with the increasing of bacterial density from 1×103 to 1×107
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cells/mL, however, the MC-LR was reduced by nearly 94% within 14 h irrespective
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of bacterial density. Measurement of extracellular and intracellular MC-LR revealed
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that the toxin was decreased by 92% in bacterial cell incubated systems relative to
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control and bacterial cell-free filtrate systems. The results confirmed that the bacterial
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metabolite caused 92% lysis of Microcystis aeruginosa cells, whereas the bacterial
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cells were responsible for approximately 91% reduction of MC-LR. The joint efforts
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of the bacterium and its metabolite accomplished the sustainable removal of algae and
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MC-LR. This is the first report of single bacterial strain that achieve these dual
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actions.
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TOC
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INTRODUCTION
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The occurrence of cyanobacterial-dominated harmful algal blooms (cyano-HABs)
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in freshwater ecosystems has become a global environmental and public health
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concern due to the production of a wide range of toxic secondary metabolites
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including cyanotoxins.1 Microcystins (MCs) are the most frequently detected
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cyanotoxins produced by a number of cyanobacteria genera and are known to cause
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liver damage, promote tumor production, and have been linked to neurological
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disorders, hence, they are highly toxic to wildlife, livestock, and human beings.2
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Because cyano-HABs and MCs present a high risk to users of infested waters,
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immediate reduction of the hazards are desirable. Several methods including the
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application of algicides and chemicals,3 mechanical collection of cyano-HABs,4 and
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flocculation of cyano-HABs cells5 have been tested. These methods are effective for
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removal of cyano-HABs, whereas damage of the algal cells may occur during the
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removal process,6 which triggers the release of cell-bound MCs.7, 8
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Some studies investigated potential measures for the removal of cyano-HABs
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without breaking algal cells. Within a specific concentration range, copper-algicides
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efficiently caused damage of Microcystis aeruginosa without releasing MCs.3 A study
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on Microcystis aeruginosa mitigation using chitosan demonstrated damage of algal
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cells, but the extracellular concentration of MCs increased slightly within certain
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periods.9 A similar result was also observed when sediment dredging and Phoslock®
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were applied in combination for the treatment of cyanobacteria and MCs.10 We
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previously proposed a method for removal of cyano-HABs and MCs using 4
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chitosan-modified soil flocculation plus capping with soil where MCs-degrading
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bacterial attached.11 However, the effect was impaired in the field since capping may
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suffered from multiple factors such as vertical transport and resuspension.12 These
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measures contributed to some extent to reduced levels of dissolved MCs or mitigation
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of MCs from the water phase to the sediment, however, only when MCs in natural
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water systems were degraded to less toxic products can the negative effects be
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diminished.11 Moreover, chemical methods were usually not environmentally friendly
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due to their side effects to aquatic ecosystems, and physical methods were usually too
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costly to apply.13 Biological approaches against harmful algal blooms were of
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particular interest in freshwater systems. Biological agents such as viruses, bacteria,
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fungi, and heterotrophic flagellates were found to play a major role in the regulation
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and termination of harmful algal blooms.14-17 To date, many algicidal bacteria had
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been isolated and applied to control algal blooms, whereas the algicidal bacteria
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isolated from natural environments resulted in the lysis of cyanobacterial cells and
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release of intracellular MCs. Therefore, it remained a challenge to find a solution that
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can simultaneously remove cyano-HABs and eliminate the released MCs in an
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effective and environmentally friendly way.
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It was found that the interactions between bacteria and toxic cyanobacteria not
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only induce the lysis of algal cells but also impact cyanobacterial physiology and
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behavior. Some recent study demonstrated that toxic Microcystis and MCs-degrading
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bacteria exhibited both direct and indirect influences on each other. For example,
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MCs-degrading bacteria induced up-regulation of the transcriptional response of the 5
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mcyD gene of toxic Microcystis and prompted the MCs production,18 demonstrating
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the natural microorganism impacted MCs production. Meanwhile, Microcystis and
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other microorganisms with varied functions coinhabited the aquatic environment. The
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coexistence of algalytic bacteria (responsible for damage of algal cells and release of
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MCs) and MCs-degrading bacteria had been proved by a previous study in which
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MCs-degrading bacteria and algicidal bacteria were isolated from heavy cyano-HAB
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water in Taihu Lake.19,20 Furthermore, in our recent study regarding MCs
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biodegradation, it was discovered that the bacterial biodegradation activities of MCs
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were inducible under certain conditions,21 implying it was possible that individual
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bacterial strains exhibiting both algicidal and MCs-degrading activities existed or can
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be cultured. However, partly due to the lack of appropriate bacterial isolation
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strategies, no research thus far had attempted to study the lysis of toxic algal cells and
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degradation of MCs by single bacterial strains in co-cultured algal/bacterial systems.
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Understanding the mechanisms will be helpful for extending our knowledge regarding
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the interaction between toxic cyanobacteria and natural microorganisms and
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providing alternatives to simultaneous cyano-HAB and MC removal.
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To cope with the knowledge gaps, in the present study, a method capable of
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isolating algal lysis and MCs degrading microorganisms was studied. Under the
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co-incubation of algae and bacteria, the morphological and quantitative responses of
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algal cells relative to the bacteria were investigated, the profile of oxygen production
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was studied, and the dynamics of MC-LR release and biodegradation were assessed.
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The objective was to study the effects of single multi-functional bacterial strain on 6
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simultaneous removal of cyano-HABs and MCs, as well as the mechanism underlying
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the dual action.
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MATERIALS AND METHODS
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Toxin-producing Microcystis and Reagents. The MCs-producing strain Microcystis
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aeruginosa FACHB905 was purchased from the Freshwater Algae Culture Collection
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at the Institute of Hydrobiology (FACHB-Collection, Wuhan, China). MC-LR
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standards were obtained from Taiwan Algal Science, Inc. (purity ≥95% by HPLC).
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Bacterial Isolation and Identification. The microorganism communities were
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collected from cyano-HABs in Meiliang Bay, Taihu Lake, China in August, 2015.
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Water samples (10 mL) were inoculated to 90 mL sterile deionized water and
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microorganisms were detached by vortexing for 30 min, subsequently, the supernatant
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was collected. The isolation of MC-degrading bacteria in the supernatant was
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performed in a mineral salt medium (MS medium) containing per litre: 112 mg
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MgSO4·H2O, 5 mg ZnSO4·H2O, 2.5 mg Na2MoO4·2H2O, 340 mg KH2PO4, 670 mg
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Na2HPO4·7H2O, 14 mg CaCl2 and 0.13 mg FeCl3, the pH of MS medium was
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adjusted to 7.0 and sterilized before use, then 5 µg/mL MC-LR was added as the sole
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carbon and nitrogen source. The medium were maintained at 25°C with shaking at
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120 rpm in the dark. To purify the culture of MCs-degrading bacteria, 200 µL samples
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of the enriched cultures were transferred and spread on a solid MS medium
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supplemented with MC-LR. On the basis of size, color, and morphology, single
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colonies isolated from the plates were selected and recognized as potential MC-LR
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degrading bacteria. Aimed at confirming the MC-LR degradation ability of the 7
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individual isolates, the pure isolates were inoculated in a MS medium in which 6.4
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µg/L MC-LR were added as the sole carbon and nitrogen source. For each treatment,
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1.0 mL of well-mixed sample was collected every day with a sterile needle and
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syringe for MC-LR analysis. The isolates that induced dramatic degradation of
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MC-LR were then used for algal lysis tests, during which the obtained bacteria were
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co-incubated with 99 mL of Microcystis aeruginosa FACHB 905 having an initial
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biomass of 2×106 cells/mL, and the mixture was cultured at 25°C under 40 µmol
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photons/(m2·sec) and a 12 h:12 h (light:dark) cycle. A negative control was tested
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using an equal volume of sterile water instead of the bacterial inoculation. Samples (1
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mL) were collected from 2 cm below the water surface, and the algal cells were
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enumerated in the counting chamber of an electromotive microscope (Axioskop 2 mot
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plus, Carl ZEISS, Germany). When the final algal cell density of Microcystis
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aeruginosa FACHB905 declined to less than 50% of the negative control, the bacteria
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were recognized as algal lysis and MCs degrading bacteria.
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For bacterial identification, the sequence of the bacterial 16S rRNA gene was
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determined and compared with the sequences available from the NCBI-BLAST
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database to obtain definitive information on the phylogenetic position. The genomic
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DNA was isolated using the Tiangen Genomic DNA Isolation kit (Tiangen Biotech
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Co., Ltd. Beijing, China). Sequencings were conducted at Sangon Biotech Co., Ltd,
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Shanghai, China. Each phylogenetic tree was obtained by comparing the sequences of
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the isolates with closely related NCBI sequences. The tree was constructed using a
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neighbor-joining analysis. 8
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Co-incubation Experiment. The isolated bacterial strain was co-incubated with
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Microcystis aeruginosa (1×106 cell/mL) in a 1000 mL BG11 medium in three groups
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with an initial bacterial density of 1×103 cell/mL, 1×105 cell/mL, and 1×107 cell/mL,
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respectively. Another flask was used as control in which only Microcystis aeruginosa
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was incubated in the BG11 medium. The four groups of cultures were made in
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triplicate and the incubation experiments were conducted under controlled laboratory
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conditions that remained constant throughout the study (25°C under 40 µmol
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photons/(m2·sec) and a 12 h:12 h (light:dark) cycle). During the incubation
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experiment, 1 mL of each culture was removed from the flasks at 2h intervals and
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were passed through 0.22 µm pore-size filters (Track-Etched Membranes, Whatman®
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Nuclepore™) for quantitative analysis of the MC-LR toxin using UPLC-MS/MS.22 At
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the beginning and end of the experiment, 2 mL solutions were collected from each of
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the four groups, then the micrographs property of the algal or bacterial cells in the
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solutions were acquired using a XL-30 scanning electron microscope (Philips Corp.,
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The Netherlands).
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In order to further investigate the response of algal photosynthesis against varied
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bacteria stresses, another experiment was conducted at a 4000 µL scale with four
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groups having the same initial Microcystis aeruginosa but varied bacterial density as
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mentioned previously. Oxygen levels were constantly measured using a Clark-type
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oxygen microelectrode (Unisense, Denmark) with a tip diameter of 25 µm, a stirring
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sensitivity of
