Environmental Pollution 266 (2020) 115283

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Environmental Pollution

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Cyanotoxin impact on microbial-mediated transformations at the interface of sediment-water column in surface water bodies

Hanyan Li a, Marielle Hollstein a, Aditi Podder a, Vedansh Gupta b, Michael Barber a, * Ramesh Goel a, a Department of Civil and Environmental Engineering, University of Utah, UT, USA b Microvi Biotech Inc, Hayward, CA, USA article info abstract

Article history: Harmful cyanobacterial blooms produce lethal in many aquatic ecosystems experiencing eutro- Received 30 April 2020 phication. This manuscript presents results on the effects of cyanotoxins on the aerobic microbial Received in revised form communities residing at the interface of sediments and water columns with the ammonia-oxidizing 23 July 2020 (AOB) as the model microbial community. -LR (MC-LR), a heavily researched cya- Accepted 26 July 2020 notoxin variant, was used as the model cyanotoxin. To measure cyanotoxin influence on the activity of Available online 6 August 2020 nitrifying microbial communities, an enriched culture of AOBs collected from an ongoing partial This paper has been recommended for nitrification-nitritation reactor was examined for its exposure to 1, 5 and 10 mg/L of MC-LR. The nitri- acceptance by Markus Hauck. tation kinetics experiment demonstrated MC-LR’s ability at 1, 5, and 10 mg/L concentrations to prevent ammonium oxidation with statistically significant differences in nitritation rates between the blanks and Keywords: spiked samples (One-way ANOVA, p < 0.05). Significantly decreased dissolved oxygen (DO) consumption Sediment interface during oxygen update batch tests demonstrated ’sinfluence on AOB’s oxidizing capabilities when Reversible kinetics exposed to even lower concentrations of 0.75, 0.5, and 0.25 mg/L of MC-LR in a separate set of experi- Microcystin-LR ments. Based on competitive kinetics, the MC-LR inhibition coefficient-the concentration needed to Nitrifiers produce half-maximum inhibition of the mixed community AOBs was determined to be 0.083 mg/L. The Kinetics m amoA gene stress tests proved the recovery of nitritation to some extent at lower MC-LR concentrations (1 and 5 g/ L), but significant irreversible inhibition was recorded when the AOB population was exposed to 10 mg/L MC-LR. The comparisons of amoA gene expressions corresponded well with nitrifying kinetics. All concentrations of MC-LR spiking were determined to produce a discernible impact on the AOB nitritation rate by either destroying the bacterial cell or immediately inhibiting the amoA gene expression. © 2020 Elsevier Ltd. All rights reserved.

1. Introduction cyanobacterial secondary metabolites, are one of the most potent contaminants found in freshwater ecosystems (Stewart et al., Over the last few decades, the growth of harmful algal blooms 2008). Human, ecological and microbial health is affected by the (HABs) in many aquatic systems has become a prevalent problem toxins produced by . Microcystin-LR (MC-LR) is due to increased agricultural runoff, wastewater treatment plant mainly a dangerous cyanotoxin, known for accumulating in the effluents and other nonpoint source discharges with high concen- of humans, livestock, and other animals (Lone et al., 2015). trations of nutrients getting into lakes (Heisler et al., 2008). While As recent studies demonstrate how climate change correlates to algal and cyanobacterial biomass occur naturally in aquatic eco- cyanobacterial blooms (Wells et al., 2015; Griffith and Gobler, systems, cyanobacterial blooms are amplified by anthropogenic 2020), cyanotoxin’s ecological impact has become a predominant activities, such as excess agricultural, point and nonpoint run offs question in relation to lake water post-bloom conditions (Dalu and (Paerl et al., 2011; Paerl and Otten, 2013). Cyanotoxins, which are Wasserman, 2018). While lake water conditions during algal blooms are toxic to the surrounding plant, animal, and microbial species, long term effects of cyanobacterial blooms are more in- fi * Corresponding author. de nite. Moreover, cyanotoxin strains have appeared throughout E-mail address: [email protected] (R. Goel). the water column, sediment, , and even tertiary plants in https://doi.org/10.1016/j.envpol.2020.115283 0269-7491/© 2020 Elsevier Ltd. All rights reserved. 2 H. Li et al. / Environmental Pollution 266 (2020) 115283 nearby agricultural plots (El Khalloufi et al., 2011; Corbel et al., 2. Materials and methods 2015a; Cao et al., 2017; Richardson and Kimura, 2017). By extract- ing (MCs) from lake sediments, researchers were able 2.1. Nitritation kinetic experiment under cyanotoxin to measure high concentrations of those at the interface of sedi- ments and water columns (Chen et al., 2006). As cyanotoxins are A mixed community of AOBs from an ongoing enrichment was introduced into the environment, the residing bacterial community used to conduct cyanotoxin toxicity experiments on biological composition experiences a shift, suggesting that the toxins may ammonium oxidation to nitrite rates and amoA gene expressions. possess control over the bacteria (Wilhelm et al., 2011; Su et al., The details of the AOB enrichment reactor can be found in Kotay 2017; Peng et al., 2017; Hampel et al., 2018). Bacterial and light et al. (2013) and this AOB enriched reactor has been in operation degradation, as well as other removal pathways, can mitigate cya- consistently over the last 8 years (Gupta and Goel, 2019). Quanti- notoxin impact on the environment (Chen et al., 2008). However, fication of the mixed AOB community in the nitritation reactor the persistence of MC-LR was even measured at low concentrations suggests Nitrosomonas europaea as the dominant species (62 ± 7% of recorded cyanobacterial biomass, indicating MC-LR’s resilience of total community). To conduct toxicity experiments, biomass to environmental degradation (Lahti et al., 1997). The duration of samples were taken from the ongoing enriched reactor and washed the toxin’s presence after cyanobacterial decay is also mostly three times with deionized water for residue nutrient removal unknown. before using it for experiments. Washed biomass was purged Within the bacterial processes, the aerobic process that converts through a 20-gauge needle to break the biomass pallet and disperse þ ð Þ it. The total suspended solids (TSS), volatile suspended solids (VSS) ammonium-nitrogen (NH4 N) to nitrites NO2 N and nitrates ð Þ fi and initial NHþ N and NO N concentrations were measured NO3 N through oxidation is referred to as nitri cation, and the 4 2 essential component of the overall N cycle (Dasgupta et al., 2020; immediately after dispersion. The bulk biomass was then spiked fi þ with 2e3 mg/L of NHþ N, well mixed and divided into eight equal Podder et al., 2020a). Partial nitri cation (nitritation), NH4 4 fi fi aliquots of 100 mL each in 125 mL acid-washed Wheaton® serum N oxidation to NO2 N, is the rst step in the two-step nitri ca- tion process mediated by autotrophic ammonia-oxidizing bacteria bottles. Of these eight serum bottles, four were spiked with a þ known concentration of MC-LR, and the rest were used as the (AOB) (Peng et al., 2017). NH4 N oxidation by AOBs is mediated through the expression of ammonia monooxygenase (amoA)asan control for four different time steps. This marked the beginning of þ enzymatic catalyst under aerobic conditions (Podder et al., 2020b; toxicity experiments and initial NH4 N and NO2 N were Rotthauwe et al., 1997). This process actively occurs at the measured at zero time. Additionally, for this study, three concen- sediment-water interface; due to in-situ aerobic conditions and trations of MC-LR (1, 5, and 10 mg/L) were used to run three different nutrient accumulation on the top of sediments (Hong et al., 2019; sets of experiments to reflect typical concentrations of MC-LR in Wu et al., 2013). Since MC-LR settles down to sediments through surface waters during algal blooms. Based on the guidelines by the water column after cyanobacterial cell death, the toxins will World Health Organization, the “low risk” category for MC has been establish contact with the sediment-water interface, especially set at 10 mg/L for recreational water, and this category was with aerobically respiring communities such as the nitrifying commonly found for algal-bloom water bodies (Lindon and community. Wind-induced agitation, water currents, and human Heiskary, 2009; Kohlhepp, 2015). The experiment sets were first activities further aid lake water mixing, thereby enhances cyano- conducted with 10 mg/L MC-LR and subsequently continued at toxin exposure and oxygen content at the water-sediment interface lower concentrations of 5 and 1 mg/L. Serum bottles were sealed (Chen et al., 2008). Regardless, factors such as MC-LR that inhibit airtight using a 20 mm butyl rubber Teflon faced septa (Fischer, microbial-mediated nitrification among AOB could potentially USA) and 20 mm aluminum crimp caps. They were aerated and disrupt nitrogen cycling within the lake environment (Beman et al., mixed continuously by purging air through a 20-gauze needle to 2011; Peng et al., 2017). However, the interactions between pre- the liquid and with another unconnected needle for the air to exit existing microbiota and cyanotoxins within the sediment- the headspace of bottles. Samples were taken from the control and interface layer have yet to be fully interpreted. Therefore, a toxin spiked set at 0.5, 1, 2 and 3 h and, the contents of each bottle þ ; detailed understanding is needed about the effect MCs could pose were tested for NH4 N NO3 N and NO2 Nconcentrations. on aerobic microbial communities participating in critical ecolog- þ NH4 N was measured using HACH Nitrogen-Ammonia Reagent ical processes and residing at the interface. Set TNT Kit (Hach, USA) and, NO3 Nand NO2 Nwere measured The overall goal of this research was to evaluate the effect of on an Ion Chromatograph (Metrohm 883 Basic IC plus) for anion selected concentrations of MCs on aerobic microbiota, potentially detection (EPA method 300) (Pfaff, 1993), respectively. Triplicate residing at the interface of sediments and the water column. We samples (each with 10 mL volume) of mixed liquor from each bottle þ selected nitritation, aerobic NH4 N oxidation to nitrite, as the were also obtained at each time step to measure TSS and VSS model aerobic process and an essential component of the overall following the USEPA method 1684. The rest of the sample volume nitrogen cycle. Early study has studied the treatment of a high remaining in the bottle was used for microbial analysis at each time density of cyanobacteria on AOBs (Peng et al., 2017). However, the step. impact of cyanotoxins on the nitrifying community alone was not adequately studied. The objectives of this study were a) to deter- 2.2. Stress study mine AOB community response from immediate exposure to microcystin by measuring and analyzing nitritation rates through Stress tests were conducted to evaluate whether MC-LR toxicity þ ’ NH4 N oxidation; b) measure the cyanotoxins residual effect on to AOBs is reversible or irreversible. Toxicity tests were conducted AOB with a stress study in the effort to facilitate our comprehension in a similar fashion, detailed in section 2.1. After 3 h of incubation of the cyanotoxins’ fate within microbial communities after their with toxins, the biomass samples that were spiked with different initial introduction. Insight is provided on how cyanotoxins are concentrations of MC-LR were washed twice with DI water to altering the AOB nitritation and oxygen uptake rates, whether the remove any residual MC-LR in the bulk solution or toxins poten- toxin is inhibiting the amoA gene expressions or irreversibly tially stuck to the biomass. The washed biomass samples were damage the bacterial cells. e þ spiked with 2 3 mg/L of NH4 N and the experiment was H. Li et al. / Environmental Pollution 266 (2020) 115283 3

þ 2.4.2. Real-time Polymerase Chain Reaction continued further to monitor the NH4 N oxidation in the absence þ ; ; Real-time Polymerase Chain Reaction (RT-PCR or qPCR) was of MC-LR. The analysis for NH4 N NO2 N, NO3 N TSS, VSS, and microbial activities were determined as detailed in section 2.1 conducted to quantify the functional ammonia-oxidizing gene, of this manuscript earlier. ammonium monooxygenase (amoA) of the AOB (Rotthauwe et al., 1997). The purpose was to normalize the ammonia-oxidizing rates or oxygen uptake rates based on functional gene copies, in addition to biomass (measured by VSS). The normalization based 2.3. Cyanotoxin inhibition kinetics evaluation on functional gene copies has been applied in previous studies (Ma et al., 2019). The primers applied to target the amoA gene were the MC-LR inhibition coefficient was estimated experimentally by amoA_1 F/amoA_2R listed in Table 1(Rotthauwe et al., 1997). All exposing the nitrifying biomass to four different concentrations reactions were carried out in a total volume of 20 mL, containing (0.25 mg/L, 0.5 mg/L, 0.75 mg/L, and 1 mg/L) of toxins in separate sets 10 mL AB Power SYBR® Green Master Mix, 1 mL of each primer of experiments. A control experiment where the biomass was not (10 mM), 5 mL of DNA template and 3 mL of nuclease-free water. subjected to any toxin was also run simultaneously. As the MC-LR Plasmids were used as a positive control and series diluted 10 times spiked AOB feed solution was aerated for approximately 30 min, using ultrapure water to the concentrations of 108 copies/5 mLto inhibition was measured as a reduction in the specific Oxygen 103 copies/5 mL. The amplification efficiency was tested to be higher Uptake Rate (sOUR) and described using a non-competitive inhi- than 80% during the entire process. Beforehand, plasmids were bition model as shown by equation (1) (Chandran and Love, 2008). cloned from reactor biomass using TOPO® TA cloning kit/TOP10 The dissolved oxygen was measured using a HACH HQ40D Portable (ThermoFisher, USA) and followed by E. coli incubation until the Multi Meter (Hach, USA). plasmids can be extracted using Zyppy® Plasmid Miniprep Kit (Zymo Research, USA). qPCR was conducted using a QuantStudio 3 ¼ sOURo sOURinh (1) Real-Time PCR system (ThermoFisher, USA) in the process of 1 þ Si Ki 2 min at 50 C, 5 min at 94 C; then 40 cycles consisting of 60 s at 94 C (denaturation), 90 s at 60 C (annealing), 90 s at 72 C Ki is the inhibition coefficient (expressed as mg/L MC-LR), (elongation), and a final cycle consisting 10 min at 72 C. All sam- sOUR is the inhibited sOUR (mg O /min), sOUR is the sOUR for inh 2 0 ples, including plasmids (positive controls), were conducted in the control solution without any MC-LR cyanotoxin (mg O /min), Si 2 triplicates. is the concentration of MC-LR (mg/L). Ki was estimated using a linear regression model as follows: 2.4.3. Gene expression 1 ¼ 1 þ Si Under cyanotoxin stress, the expressions of amoA were also * (2) sOURinh sOURo sOURo Ki relatively quantified. The gene expression experiment was tested in a QuantStudio 3 Real-Time PCR system (ThermoFisher, USA) using the DDCT method, developed by Schmittgen and Livak (2008). The equation was detailed below, and relative quantification (RQ)was DD calculated as 2 CT . The RQ of reference sample was normalized to 2.4. Microbial analysis 1 based on this method. 2.4.1. DNA and mRNA extraction DDCT ¼½ðCT ðtarget geneÞ Bacterial DNA and RNA were extracted from AOB biomass from C ðinternal control geneÞSample A½ðC ðtarget geneÞ both the blank and MC-LR spiked mixed liquors under different T T ð Þ conditions. Biomass was concentrated by filtering through a CT internal control gene Reference Sample 0.45 mM filter paper (Millipore, USA). DNA was extracted using a (3) PowerSoil® DNA Isolation Kit (Qiagen, Germany). RNA was ob- tained using a PureLink® RNA mini kit (Life Technology, USA). Following RNA extraction, residual genomic DNA was removed from total RNA using an on-column PureLink DNase set (Life - CT represents the number of cycles taken for the fluorescent Technologies, USA). The concentrations and quality of DNA and RNA signal of the reporter dye to cross an arbitrarily placed were checked using Nanodrop 2000c (ThermoFisher, USA) and threshold. stored at 80 C. To be specific, samples with a 260/280 ratio of - DDCT represents the comparative CT method. It is calculated around 1.8 (pure DNA) and 2.0 (pure RNA) were selected for based on the above equation to compare the expression level downstream analysis. After the extraction of RNA, reverse tran- difference between control and treatment samples, as well as scription was immediately conducted to convert 0.4 mgRNAto normalizing target genes based on the internal control genes. cDNA in each sample using the SuperScript® VILOTM cDNA syn- thesis kit (Life Technology, USA). They were further used as tem- In this study, the sample extracted at t ¼ 0 in each batch study plates for gene expression analysis. was used as the reference sample for other time steps (0.5 h, 1 h,

Table 1 Primers used in qPCR and gene expression analysis.

Target Primers Sequences References

amoA gene amoA_1F GGGGTTTCTACTGGTGGT Rotthauwe et al. (1997) amoA_2R CCCCTCKGSAAAGCCTTCTTC AOB 16S gene CTO 189f A/B GGAGRAAAGCAGGGGATCG Hermansson and Lindgren (2001) CTO 189f C GGAGGAAAGTAGGGGATCG RT1r CGTCCTCTCAGACCARCTACTG 4 H. Li et al. / Environmental Pollution 266 (2020) 115283

2 h, and 3 h). Within each sample, the CT for the functional gene oxidizing bioreactor, the presence of nitrite oxidizers was not ex- (amoA gene) was obtained and normalized by the 16S rRNA gene pected, and it was confirmed through periodic nitrate measure- copy numbers of AOB (the internal control gene). The amoA gene ments. NO3 N concentrations in serum bottles were found to be and 16S rRNA gene of AOB in samples were targeted by primers below detection limits. The first set of experiments was conducted amoA_1 F/amoA_2R (Rotthauwe et al., 1997) and CTO 189f (A/B/C)/ with 10 mg/L MC-LR to evaluate the effect of the toxin on nitrifying RT1r (Hermansson and Lindgren, 2001), respectively. Primers biomass. The results are plotted in Fig. 1 in different panels for applied are listed in Table 1. Two master mix solutions were created NHþ N increase (panel A) and NO N decrease (panel D) for m ® 4 2 for each primer set, in which 10 L AB Power SYBR Green Master 10 mg/L MC-LR concentration. As evident from panel A in Fig. 1, m m m þ Mix, 1 L of each primer (10 M), 1 L of DNA template and absolutely no oxidation of the NH N was recorded at the 10 mg/L nuclease-free water were added to make a total volume of 20 mL. 4 MC-LR concentration. On the other hand, noticeable nitrification The qPCR process was modified to target both the amoA gene and þ activities were recorded with a consistent decrease in NH N the AOB 16S rRNA gene using the VeriFlex temperature control 4 with a corresponding increase in NO N concentrations in the mode during the annealing process. It was followed by 2 min at 2 fi 50 C, 5 min at 94 C; then 40 cycles consisting of 60 s at 94 C control experiment (panel D in Fig. 1). Signi cant differences were found among nutrient concentration changes between controls and (denaturation), 90 s at 56 C for amoA gene and 60 C for AOB 16S m < rRNA gene (annealing using VeriFlex mode), 90 s at 72 C (elon- 10 g/L MC-LR spiked batch tests (One-way ANOVA, p 0.05). With the observation that 10 mg/L toxin concentration caused gation) and a final cycle consisting 10 min at 72 C. The experiment fi was conducted in triplicates. The average, max, and min RQ values complete inhibition of nitri cation, subsequent batch tests were conducted with lower MC-LR concentrations of 5 mg/L and 1 mg/L. were plotted for each batch of samples in the results section. þ The results of these experiments for NH4 N concentration are plotted in panels B and C in Fig. 1. Surprisingly, the mixed com- 2.4.4. Live and dead analysis þ munity of AOBs did not exhibit any NH4 N oxidation at 5 and To evaluate the lethal effects of MC-LR, live and dead analysis m was conducted to have a qualitative estimation of the membrane 1 g/L MC-LR concentrations, demonstrating severe inhibition of m þ integrity at population level after the exposure to 1, 5, and 10 mg/L of nitritation at even 1 g/L. On the other hand, the NH4 N oxidation MC-LR during a 3-h period. In this experiment, bacterial cells were in corresponding control batch tests shown in panels E and F of stained with light-sensitive BacLight® dyes containing Propidium Fig. 1 is evident and convincing with corresponding increases in Iodide and SYTO 9 nucleic acid stain (ThermoFisher, USA), and were NO2 N concentrations. The live and dead analysis further depic- examined under an Olympus® BX51 light microscope (Olympus, ted the deadly effects of MC-LR at 1, 5 and 10 mg/L concentrations. USA). 10 mg/L of Propidium Iodide and 10 mL SYTO 9 master stocks However, it is also evident from Fig. S1 that not all bacteria are were combined to create a working stock in the dark. Afterward, killed even at 10 mg/L (Fig. S1). In most conditions, MC 2 mL of the working stock was mixed with 198 mL of Ultra-Pure concentrations are between 1 and 10 mg/L in surface waters (Song Water to create the master mix. Next, a 0.22 mm polycarbonate et al., 2007; Liu et al., 2011; Preece et al., 2017; Turner et al., filter (GVS Filter Technology, UK) with filtered AOB biomass on it 2018). While the World Health Organization recommends was placed onto a glass slide and dabbed with Kim wipes to remove limiting microcystin concentration to 10 mg/L in water for recrea- excess water. The filter was left in place for 3e5 min and later tion purposes (Bartram and Chorus, 1999), the effects of MC-LR on removed with a pair of tweezers. A plastic well was placed on the AOB are visible at significantly lower concentrations in this study. microscope slide to encompass the removed filter area, and 200 mL It is observed that the direct exposure of the AOB population to of the master mix was added inside the well. The mix was left on MC-LR at 1 mg/L concentration has similar inhibition effects as the slide in a dark room for 20 min. Later, the plastic well was 10 mg/L. Additionally, in all treatment spiked with MC-LR, there þ removed with tweezers, and the area was cleaned with Mili-Q was, in fact, a slight increase in NH4 N concentrations. This was water. One drop of Dabco®33-LV solution (Sigma-Aldrich, USA) surprising as well as un-explanatory at this point. Microcystins was added to the area and covered with a micro cover glass, sealed could introduce variability to nitrogen transformations occurring and placed under the microscope with 100x magnification. within freshwater ecosystems, and N removal rates have been more unpredictable in bloom-dominated ecosystems (Peng et al., 2017). 2.5. Statistical analysis One possible reason could be the occurrence of dissimilatory þ reduction of NO2 N to NH4 N(Bu et al., 2017). As the reduction One-way ANOVA was applied to detect the changes in nutrient of functional transcripts under the toxicity could be one of the main concentrations between blank and treatment groups for each set of factors causing inhibitory nitritation rates (Kapoor et al., 2016b; kinetic and stress studies using Rstudio software v3.5.1 (Team, Kim et al., 2016), some other genes may be simultaneously acti- 2013). The variations of MC-LR concentrations on amoA gene ex- vated during the nitrification inhibition as bacteria tried to acquire pressions at the kinetic and stress status were also compared using nutrients from the surrounding environment. The corresponding fi one-way ANOVA. Signi cance was determined at an alpha level of decreases in NO2 N concentrations were also recorded in toxin 0.05 (p 0.05). SigmaPlot v 10.0 from Systat Software, Inc., San Jose spiked samples with respect to NO2 N concentrations present at California USA, was applied for plotting the figures. the beginning of experiments. On the other hand, a consistent þ decrease in NH4 N with a corresponding increase in NO2 N 3. Results and discussion were recorded in all un-spiked (control) samples. However, the DNRA theory remains speculative at this time, and perhaps similar 3.1. Effect of MC-LR on nitrification experiments in the future would benefit from the simultaneous measurement of DNRA activity. Enriched biomass comprised mostly of AOBs was subjected to In the control bottles which were not spiked with MC-LR, the þ NHþ N oxidation rates were 0.68 ± 0.16, 0.17 ± 0.17, 0.54 ± 0.19 different concentrations of MC-LR and, decreases in NH4 N and 4 and 0.57 ± 0.11 mg/L in three different control bottles tested be- increases in NO2 N concentrations were recorded as a function of time. Since the biomass was obtained from an enriched ammonia- tween time periods of 0e0.5, 0.5e1, 1e2 and 2e3 h, respectively. H. Li et al. / Environmental Pollution 266 (2020) 115283 5

Fig. 1. (AeC) NH4 N concentrations versus time at 1, 5, 10 mg/L MC-LR respectively and (DeF) NO2 N concentrations versus time at 1, 5, 10 mg/L MC-LR in nitrification kinetics study. Each data point is based on triplicate measurements.

The corresponding increases in NO2 N concentrations were on nitrifying bacteria were lethal/irreversible or reversible. This 0.35 ± 0.37, 0.44 ± 0.13, 0.61 ± 0.18 and 0.33 ± 0.37 mg/L, respec- question is important as it will inform whether the nitritation ac- tively. With normalization, the 3-h nitritation rates were measured tivity would restore to its normal conditions after algal blooms have as 0.0035 ± 0.0009 gNgVSS 1hour 1 and 7.44 ± 2.38E-07 occurred and cyanotoxins have degraded. To evaluate whether the mgL 1copy 1hour 1 for the control group. Since the nitrifying effect was inhibitory, e.g., reversible, batch tests were further biomass was obtained from an ongoing reactor primarily oxidizing conducted with nitrifying biomass that was first subjected to MC- þ fi LR exposure and then washed twice to get rid of any residual toxin. NH4 N to NO2 N (e.g., nitritation), no signi cant change of The NHþ N and NO N concentrations with different NO3 N was measured in the blank or sample bottles. This 4 2 established the fact that nitritation achieved exclusively from AOB exposed MC-LR concentrations are plotted in Fig. 2. Similar to the activity. Above all, there were no significant nitritation activities nitritation kinetics experiment, all three samples in the stress study detected for the toxin-spiked group, demonstrating that MC-LR initially experienced an inhibited nitritation rate, which demon- fi þ was severely toxic/inhibitory to nitri ers even at low strates the lasting effect of the MC-LR (Fig. 2). As expected, NH4 N concentrations. decreased and NO2 N increased in control batch tests. Surpris- While other researches have corresponded the low DO con- ingly, a trend of NHþ N decrease and NO N increase was also fi 4 2 centrations with inhibited nitri cation rates and high ammonium observed for groups in which the nitrifying biomass was previously fl ux in sediment (McCarthy et al., 2008; Abell et al., 2011), the exposed to 1, 5 and 10 mg/L MC-LR but reused after thoroughly þ NH N oxidation in our study was connected with a relatively þ 4 washing with DI water (Fig. 2). It appears that the rate of NH4 N high DO concentration throughout the experiment. Therefore, in oxidation in sample bottles, in which the biomass was originally this research, limited oxygen levels may not likely be a factor exposed to the toxin, was slower than the rates in their corre- þ impeding AOB activity, as the NH4 N concentrations in control sponding control batch tests (One-way ANOVA, p < 0.05). These batch tests decreased by 45e60% in comparison to spiked sample results demonstrate that cyanotoxin toxicity is reversible to some batch tests (discussed later). þ degree. Higher nitritation rates (changes in NH4 N and NO2 N concentrations) were detected at 1 and 5 mg/L but not at 10 mg/L. 3.2. Nitritation recovery potential after MC-LR spiking Especially in bottles where biomass was spiked with MC-LR and þ washed, the NH4 N decreases were 0.80, 1.00, and 0.53 mg/L at 1, The direct spiking experiments clearly indicated that nitritation 5, and 10 mg/L exposure between periods of 0e3 h, respectively. was severely inhibited with MC-LR concentration as low as 1 mg/L. With normalization, the 3-h nitritation rates were estimated to be 1 1 From batch experiments, it was not clear whether the toxin effects 0.0015, 0.0019 and 0.0010 gNgVSS hour and 1.71E-06, 2.47E-06 6 H. Li et al. / Environmental Pollution 266 (2020) 115283

Fig. 2. (AeC) NH4 N concentrations versus time at 1, 5, 10 mg/L MC-LR respectively and (DeF) NO2 N concentrations versus time at 1, 5, 10 mg/L MC-LR in nitritation stress study. Each data point is based on triplicate measurements.

and 3.82E-07 mgL 1copy 1hour 1 for the group exposed to 1, 5 and nitritation study in this study, a rise in the relative abundance of the 10 mg/L, respectively. These results demonstrate that the nitrifica- amoA gene was also observed when the AOB containing soil was tion inhibition by the model cyanotoxin is reversible after con- irrigated with low concentrations of MC-LR for a 14-day period in tacting with MC-LR for 3 h at some optimum MC-LR concentrations. the previous study (Corbel et al., 2015b). Moreover, after a mod- AOBs were speculated to recuperate from the initial MC-LR spiking erate density of cyanobacteria (106 cells/L) dominated by Micro- at lower concentrations, which was also proved by the gene cystis was introduced to an AOB community in a laboratory expression analysis for an increased nitritation potential (relatively microcosm, the amoA gene expression values were determined to higher gene expression) after exposure to 1 and 5 mg/L MC-LR (Fig. 4 be greatest relative to the high-density cyanobacteria (108 cells/L) D, E). The lower concentration (1 mg/L and 5 mg/L) may show better and untreated microcosm (Peng et al., 2017). The resilience of AOB recovered nitrifying activities after exposure. This indicates that the is similarly depicted through nitrification recovery after exposure presence of toxins during heavy harmful algal blooms (HABs) will to metal (Ruyters et al., 2013). Another study evaluating bacterial affect ecosystem functioning by interfering with important pro- resilience exposed microbial communities to heat stress over an cesses such as nitrogen cycling. However, this negative effect is extended period of time also demonstrated the AOB’s increased reversible to some extent meaning that the ecosystem functioning nitrification potential rates (Epelde et al., 2014). All of the above can be restored once the bloom is disappeared and toxins are analyses suggested that microcystin exposure may induce greater degraded. Previous studies also demonstrate that the ecosystem amoA gene expression in AOBs even if their nitrification potential is function can usually be restored by the degradation of cyanotoxins low. after blooms have disappeared (Koreiviene_ et al., 2014; Rastogi In contrast, the addition of relatively high concentrations of MC- et al., 2015). AOB recovery may be a direct response to ambient LR (10 mg/L) caused crushing effects on the AOB’s cellular mem- þ brane and reduced the amoA gene expression levels due to the cyanotoxin levels, as well as the level of NH4 N present in the solution; these conditions indirectly impact the microbial physi- direct killing effect (Fig. 4 F and S1). Previous studies also reported ology, functions, and community composition (Giaramida et al., that the harmful effect is visible when exposed to relatively higher 2013). concentrations of MC-LR at a regular frequency. For example, the Similar to our study, the resilience of AOBs was also indicated by significant inhibitory effects were previously observed with the previous studies, albeit in the soil environment. When evaluated treatment of a high density of cyanobacteria on AOB (Peng et al., through a 90-day trial, the nitrification potential of soils irrigated 2017). This also suggests cyanotoxin’s immediate effect on AOB with 50 and 100 mg/L of MC-LR experienced similar N trans- cultures and long-term adverse effects at a high density of cyano- formations trends compared to the control (no toxin applied) while bacteria. While AOB is recognized to recover at lower concentra- soils containing relatively lower MC-LR (5e20 mg/L) concentrations tions of MC-LR exposer and as observed in this study as well, other experienced higher nitrification potentials (Corbel et al., 2015a). studies analyzing the effects of MC contaminated irrigation on Just as amoA gene expression ultimately increased in the stress overall plant morphology demonstrated a decreased quality and H. Li et al. / Environmental Pollution 266 (2020) 115283 7 yield of crops with regular exposer to 10 mg/L of MC-LR or higher (inhibition coefficient ¼ 17.29 mg/L) or (inhibition (Lee et al., 2017; Zhu et al., 2018). The cyanotoxin may reduce the coefficient ¼ 0.12 mg/L) towards ammonium inhibition (Kapoor harvest by affecting the N cycle of the soil bacterial communities. It et al., 2016a, 2016b). was also found that high concentrations of MC-LR (>10 mg/L) significantly decreased soil potential nitrification rate, together 3.4. Ammonium monooxygenase (amoA) gene expressions with the decline of amoA gene abundance and these past results are very much in agreement with our study where we also used 10 mg/L In all batch studies, the relative quantification of the AOB’s amoA MC-LR to study inhibitory effects on AOBs. gene was obtained through the DD Ct method for spiked samples and controls at different sampling time periods. Basically, the gene expressions (control and treatment groups) at different sampling 3.3. Determination of toxin inhibition coefficient points is relatively quantified based on time 0 (RQ ¼ 1 for time 0), so that control and treatment groups are comparable at each sampling MC-LR toxicity to nitrifying activity was also evaluated by con- point. The higher RQ demonstrates relatively higher gene expres- ducting oxygen uptake tests with MC-LR spiked in batch tests. The sion activities. The relative quantification (RQ)ofamoA gene for results of the oxygen uptake rate experiments complement the AOBs in direct contact nitrification study is shown in Fig. 4 A-C. In inhibited NHþ N oxidation in the nitritation batch experiments. 4 general, comparable to the control, MC-LR spiked samples experi- fi While the nal DO concentration in the control experiment where enced a lower average RQ of the amoA gene at all measured time for the AOB biomass was not spiked with MC-LR decreased to 55% of samples containing 5 and 10 mg/L of MC-LR (Fig. 4 B, C). These re- the initial dissolved oxygen concentration due to active nitrifica- þ sults corresponded with the suppression of NH N oxidization tion, the DO concentration mostly remained unchanged in 1 mg/L 4 measured (Fig. 1B and C). The RQ values for the amoA gene are more MC-LR spiked tests throughout the 30-min experimental period variable in the samples containing 1 mg/L than the other samples indicating inhibited AOB activity. Furthermore, amoA was down- having different concentrations (Fig. 4 A). Especially, the solution regulated at 5 and 10 mg/L MC-LR concentrations correlating with containing 1 mg/L of MC-LR observed relatively higher average RQ low or no ammonium oxidation at these spiked concentrations values compared to control for the first 2 h, although the oxidation (Fig. 4 B, C). rates were suppressed (Fig. 1A) and do not correlate with the gene For five sets of experiments (0, 0.25, 0.5, 0.75, and 1 mg/L MC-LR), expression here. It is predicted that the AOBs may still try to recover VSS and amoA gene copy numbers were in the range of 60e373 mg its function under 1 mg/L MC-LR concentration by enhancing gene VSS/L and 1.60Eþ06 to 3.08Eþ06 gene copies per mL of mixed li- expressions. Except for a few outliers, the highest RQ was observed quor respectively. Oxygen uptake rates are normalized based on at time 0.5 or 1 h and decreased afterward in all batches. amoA gene copy numbers for accurate estimation. As the concen- During the stress study, the RQ values of the amoA gene are also tration of MC-LR increased, the oxygen uptake rates decreased highlighted for controls/samples, and differences in expression simultaneously. Normalized specific oxygen uptake rates (sOUR) 1 1 levels are depicted (Fig. 4DeF). The stressed nitritation study’s gene were 4.45E-08 mg O2 min copy under non-spiked conditions 1 expression procedure indicated greater RQ values for the samples (e.g. MC-LR ¼ 0 mg/L) and decreased to 1.33E-11 mg O2 min exposed to 1 and 5 mg/L of MC-LR compared to their respective copy 1 as the MC-LR dosing increased to 1 mg/L. There was no controls, as bacteria work on recovering their functions (Fig. 4 D, E). significant decrease in sOUR once the spiked MC-LR concentration However, the sample exposed to 10 mg/L yielded a relatively low exceeded 1 mg/L. The inhibition coefficient was calculated from the gene quantification compared to the control and other spiked MC- normalized DO concentrations and was determined to be 0.083 mg/ LR concentrations in the stress study (One-way ANOVA, p < 0.05) L MC-LR based on the linear regression with a coefficient of (Fig. 4 F). It is surprising to see that the gene expression was even regression to be 0.87 (Fig. 3). Although complete inhibition was enhanced after MC-LR exposure and recovery at lower concentra- observed at 1 mg/L, inhibition effect occurred at even lower MC-LR tions (1 and 5 mg/L). It may indicate that the stress response was concentrations. It also indicated MC-LR’s severe inhibitory effects activated under adverse conditions, and some gene expressions compared to heavy metals, such as Cd (inhibition were upregulated afterward (Qin et al., 2018). At the higher con- coefficient ¼ 0.76 mg/L), Ni (inhibition coefficient ¼ 1.9 mg/L), Zn centration (10 mg/L), lethal or irreversible effects may have occurred and made the AOB activity irreversible. These results well corre- sponded with the relatively higher recovered nitritation kinetics detected after exposure to 1 and 5 mg/L MC-LR when compared with 10 mg/L.

3.5. Live and dead analysis

Live and Dead Analysis was conducted to detect the lethal ef- fects caused by different concentrations of MC-LR and have a qualitative study of toxin effects on the bacterial population. Fig. S1 provided examples of live and dead analysis conducted in the study; cells staining with green and red were considered to be live and dead, respectively. The vast majority of the bacterial cells exposed to only 1 mg/L of MC-LR seem viable. However, relatively larger percentages of the AOB cultures exposed to 5 and 10 mg/L of MC-LR experienced cell death or destroyed cellular membranes, as indicated by the red or orange illumination. It is noticeable that lethal or membrane destroy may occur under all applied concen- m Fig. 3. Specific Oxygen Uptake Rate (sOUR) of AOB in feed solution exposed to 0, 0.25, trations. However, even the set with 10 g/L contact for 3 h does not 0.5 and 0.75 mg=L of MC-LR. seem to kill all bacteria, instead, to inhibit their functions. The 8 H. Li et al. / Environmental Pollution 266 (2020) 115283

Fig. 4. Gene expression levels of AOB blanks and samples spiked with 1, 5, and 10 mg=L of MC-LR, respectively. (AeC) Nitrification kinetics experiment with 1, 5, and 10 mg= L of MC- LR and (DeF) the stress study with 1, 5, and 10 mg=L of MC-LR. quantitative analysis of the exact percentage of the population live the entire ecosystem are highly influenced by cyanobacterial or dead under each concentration was not conducted in the study. blooms (Gardner and McCarthy, 2009). Nevertheless, AOB growth is þ highly dependent on NH4 N concentrations, signifying nutrient 3.6. Implications of MCs effect of harmful algal blooms concentrations, microbial composition, and toxin levels’ consider- able influence on them (Verhamme et al., 2011). Regardless, factors MC-LR’s immediate impact on AOB activity may also relate to such as MC-LR that inhibit microbial-mediated nitrification among factors that promote dominance and toxin productionin AOB could potentially disrupt nitrogen cycling within the envi- freshwater environments (Reeders et al., 1998; Gilbert (2017); ronment (Beman et al., 2011). Hampel et al., 2018). MC-LR’sinfluence on AOB in lake sediments The stress study may be well applied to predict the effects on may vary depending on in situ environmental conditions. To nitrifying activities after the ambient cyanotoxin being degraded. specify, DO, pH, ammonium, and nitrate concentrations are highly Cyanobacteria bloom may have significant effects on the ecosys- correlated with Microcystis abundance and toxin-producing ca- tem’s nitrogen cycle through the effects on cell physiology and gene pacity, further altering the given ecosystem’s microbial diversity expression activities (Gardner and McCarthy, 2009; Giaramida (Raven et al., 1992; Wilhelm et al., 2011; Giamarida et al., 2013; Su et al., 2013). Results from the nitritation kinetics, stress study, and et al., 2017; Dalu and Wasserman, 2018; Li et al., 2019). Predomi- gene expression analysis further confirmed that the amoA gene nant microcystin-producing cyanobacteria, such a Microcystis sp., could exhibit visible differences in expression rates between con- are incapable of fixing N2 and prefer to feed on other forms of ni- trols and samples within a laboratory setting. Although experi- trogen (Monchamp et al., 2014; Shan et al., 2019). Still, most cya- mental results mostly correlate the abundance of the amoA gene nobacteria (including Microcystis) rely on the regeneration of with the level of nitrification and demonstrate that the quantity of ammonium to help preserve their own bacterial-bloom conditions amoA gene expressed may possess a measurable role in nutrient (Lee and Cho, 2006; Hampel et al., 2018), suggesting toxin’s inhi- transmission within natural environments, the functional gene bition of nitrification can serve as a mechanism to compete with expression abundance does not always directly reflect processes in other ammonia-oxidizers. While cyanobacteria have the capacity to lake ecosystems (Hampel et al., 2018). Exceptions could be when uptake varying forms of nutrients at lower concentrations, they can bacteria upregulated the functional gene expression, while the also survive in more extreme environmental conditions compared function was still inhibited by toxicity. It could be similar to what to their microbial counterparts (Gilbert et al., 2016). In contrast to we found when the mixed liquor directly contacted with 1 mg/L MC- cyanobacteria, other bacterial species are usually more affected by LR. Confounding factors within the laboratory setting may have also the availability and changing of N forms. Although it is established deviated from typical nitrifying community conditions found in that nitrogen transformations occur rapidly near the sediment- lake sediments due to different AOB community composition (Wu þ et al., 2013). water interface, NH4 Nand NO2 N concentrations throughout H. Li et al. / Environmental Pollution 266 (2020) 115283 9

It further established that the degradation or remediation of Declaration of competing interest MCs would benefit and boost the bacterial community restoration based on our study. While there is limited research on the fate of The authors declare that they have no known competing cyanotoxins, microcystin biodegradation in the water column, and financial interests or personal relationships that could have the sediment-interface can be considered a significant removal appeared to influence the work reported in this paper. pathway for the toxin (Chen et al., 2008). Particular species of bacteria (Sphingomonas sp., Arthrobacter sp., Brevibacterium sp.and Acknowledgement Rhodococcus sp.) occupying the same sediment as Microcystis can biodegrade these toxins (Chen et al., 2008; Ho et al., 2010; This study was partially funded through the United States Giaramida et al., 2013). Yet, cyanotoxin exposure to other bacterial Environmental Protection Agency’s STAR Grant (# 83586601) communities is mostly overlooked. As MC-producing cyanobacteria Mechanism with the project Title “Prediction of nonlinear climate proliferate, their influence on nutrient transmissions may produce variations impacts on and ecosystem processes in more predominant changes to freshwater ecology, comparable to urban and urbanizing watersheds. The views presented in this what other species are doing to mitigate the toxin problem (Song manuscript are those of authors and not necessarily reflect on the et al., 2007; Hampel et al., 2018). funding agency. While cyanotoxin concentrations were not measured over the course of the experimental procedures, MC-LR is determined to Appendix A. Supplementary data persist in sediment several weeks after the decomposition of the cyanobacteria (Lahti et al., 1997). It is acknowledged that biodeg- Supplementary data to this article can be found online at radation helps minimize MC accumulation; however, MC concen- https://doi.org/10.1016/j.envpol.2020.115283. trations are still known to exceed risk thresholds as they bioaccumulate in sediment, soils, and crops (Corbel et al., 2015a; References Levizou et al., 2017). A study measuring the accumulation of microcystin in the terrestrial soils determined that soils exposed to Abell, G.C., Banks, J., Ross, D.J., Keane, J.P., Robert, S.S., Revill, A.T., Volkman, J.K., 2011. fl 1, 10, 100, and 1000 mg/L of MCs from lake water accumulated Effects of estuarine sediment on nitrogen uxes and ammonia oxidizer gene transcription. FEMS Microbiol. Ecol. 75 (1), 111e122. equivalent MCs concentrations in dry soil samples (Cao et al., 2017). Bartram, J., Chorus, I. (Eds.), 1999. 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