Biodegradation and detoxification of saxitoxin by Bacillus flexus in batch experiments

doi:10.21203/rs.3.rs-1678858/v1

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Biodegradation and detoxification of saxitoxin by Bacillus flexus in batch experiments

https://doi.org/10.21203/rs.3.rs-1678858/v1

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Saxitoxins (STXs) are carbamate alkaloid neurotoxins produced by some species of cyanobacteria. They are water soluble and relatively stable the natural environment, and thereby represent a risk to animal and human health through a long-time exposure. STXs cannot be sufficiently removed by conventional water treatment methods. Therefore, this study investigates the potential STX biodegradation by bacteria as a promising method for toxin removal. STX biodegradation experiments were conducted using Bacillus flexus SSZ01 strain in batch cultures. The results revealed that SSZ01 strain grew well and rapidly degraded STX, with no lag phase observed. STX biodegradation by SSZ01 strain was initial-toxin-concentration–dependent. The highest degradation rate (10µg STX L^-1 day^-1) was obtained at the highest initial toxin concentrations (50µg L^-1) and the lowest (0.06µg STX L^-1 day^-1) was recorded at the lowest initial concentration (0.5µg L^-1). STX biodegradation rate increased with temperature, with highest occurred at 30 ºC. This rate was also influenced by pH, with highest obtained at pH8 and lowest at higher and lower pH values. HPLC chromatograms showed that STX biodegradation peak is corresponding to the least SXT analogue (disulfated sulfocarbamoyl-C1 variant). The Artemia-based toxicity assay revealed that this degradation byproduct was nontoxic. This suggests the potential application of this bacterial strain in slow sand filters for cyanotoxin removal in water treatment plants. Being nontoxic, this byproduct needs to be assayed for its therapeutic effects towards neurodegenerative diseases.

Artemia assay

Bacillus

cyanotoxins

degradation

saxitoxin

Saxitoxins are a group of neurotoxins produced by cyanobacteria and dinoflagellates in marine and freshwaters (Burford et al.2016; Mohamed 2018). They are carbamate alkaloids (MW=299), that block sodium channels in nerves and muscle cells and prevent electrical impulses transmission, with eventual respiratory failure and death, particularly at high toxin levels (O'Neill et al., 2016). STXs are produced by cyanobacteria species belonging to the genera Aphanizomenon, Dolichospermum (formerly Anabaena), Lyngbya, Planktothrix, Raphidiopsis (formerly Cylindrospermopsis) and Scytonema (Christensen and Khan 2020). STX is water soluble (Trainer and Hardy, 2015; Kamp et al., 2016), and stable at pH3 with exponential decease at pH7-9 (Pereira et al., 2004). STXs have been detected in drinking water [Etheridge 2010; Merel et al.2013], and thereby presenting a threat to human upon consumption of such contaminated water. Therefore, the drinking-water guideline values for STX 3μg/L has been established (WHO 2020). Conventional methods in drinking water treatment plants can sufficiently remove intracellular toxins (i.e., cells with associated toxins), but are ineffective for removing extracellular/dissolved toxins (Merel et al.2013). Additionally, removal of cyanotoxins by chemical methods can produce secondary byproducts that damage the environment (Nishu et al. 2019). As a result, an efficient cost-effective and eco-friendly methods of cyanotoxin elimination are required. In this respect, it has been reported that biodegradation is cost effective method with low risk for removing cyanotoxins from water (Mohamed and Alamri 2012; Li et al., 2015; Lezcano 2016; Mohamed et al. 2022). However, little is known about biodegradation of STXs by bacteria, compared to microcystin and cylindrospermopsin. STXs were degraded by bacteria present in bioactive filler beds in treatment plant and transformed to more toxic analogues (Kayal et al., 2008). A study by Donovan et al. (2008) has also demonstrated the biodegradation of STXs by seven unidentified marine bacteria, isolated from the digestive tract of blue mussels. Taken that Bacillus flexus was referred as an environment-friendly bioagent for elimination of arsenic and other pollutants from wastewater (Jebeli et al. 2017) and its capability for degradation of microcystin and cylindrospermopsin toxins (Alamri 2012; Mohamed et al. 2022), it could also have the potentiality to degrade STXs in aquatic environment. Hence, the present study adopted the evaluation of biodegradation capability of STXs by Bacillus flexus SSZ01 strain isolated from decayed cyanobacterial blooms in a eutrophic Saudi lake (Alamri 2010). Furthermore, our study also investigated the effects of temperature and pH on STXs biodegradation by this this bacterium, where STXs can be found under changeable conditions of pH and temperature in the natural environment.

Materials

Bacillus flexus SSZ01 strain was previously isolated from decayed cyanobacterial blooms in eutrophic lake in Saudi Arabia. It was identified by 16S rRNA gene analysis and deposited in the Gene bank with an accession of GU112451 (Alamri 2012). The strain was found to degrade other cyanotoxins such as microcystin and cylindrospermopsin (Alamri 2012; Mohamed et al. 2022). STX was purchased from Abraxis (Warminster, PA, USA). The toxin was dissolved in a small volume of sterilized lake water, and the solution was diluted to give final concentrations of 1, 5, 10, 20, 50 and 100μg L^-1.

Bacterial degradation of saxitoxin

Strain SSZ01was re-cultured for 48 hours in liquid nutrient broth (NB) medium at 30°C while shaking (120 rpm). Bacterial pellets were collected by centrifugation (6000xg, 15 min, 4°C). The pellet was washed twice with sterile 0.02 M sodium phosphate buffer (pH=7.2), re-suspended in sterilized water to the required concentration of bacterial cells and assigned as viable bacteria. Another portion of pellets was heat-inactivated by boiling at 95 °C for 1h and centrifuged (6000xg, 15 min). The supernatant was discarded and non-viable bacteria were used for potential STX adsorption. Viable bacterial cells of strain SSZ01at a final concentration of 10⁵ cfu ml^-1 was inoculated into a 250-ml sterile conical flask containing 100 ml of filtered (0.2 µm) and autoclaved lake water (pH7) and different concentrations of STX (0.5, 1, 5, 10, 20, and 50 μg L^-1). These concentrations were chosen based on the range (1 –26 μg L^-1) of environmentally relevant concentrations of extracellular STXs (Munoz et al. 2021; Li and Persson 2021). Flasks containing lake water and 100 µg L^-1STX without bacterial cells were used as control. Control and treated and flasks were incubated in a shaker (140 rpm) at 30°C. Subsamples (2ml) were aseptically taken from each flask at daily intervals for 10 days for toxin determination and bacterial growth. Bacterial growth was estimated as optical density (OD) at 600 nm wavelength. All experiments were performed in triplicate.

Effect of temperature and pH on STX biodegradation

To investigate the effect of temperature on STX degradation by strain SSZ01, bacterial cells were grown in lake water with 50µg L^-1 STX and incubated at different temperatures (10, 20, 25 and 30ºC). The effect of pH on STX biodegradation was studied by cultivation of strain SSZ01in lake water with 50µg L^-1 STX with different pH levels (6, 7, 8, 9 and10) adjusted by phosphate buffer (0.1M).

Saxitoxin analysis by ELISA

Concentrations of STX remaining in the cultures (i.e., not-degraded) during biodegradation experiments, were determined by centrifugation (6000xg, 10 min, 4ºC) of subsamples withdrawn from bacterial cultures at real time. STX concentrations were then directly determined in the supernatants using the STX ELISA kit (Abraxis, Warminster, PA, USA) according to the manufacturer’s instructions. The absorbance of the colorimetric product of antibody-conjugated enzymes was read at 450 nm on a Synergy™ hybrid multi-mode microplate reader (BioTek, USA). STX quantity was calculated from calibration curve of semi-log relationship between relative absorbance and toxin concentration using STX standard provided with ELISA kit. Each sample was run in duplicate for each assay. Detection limit for STX was 0.02 μg L^-1. The STX biodegradation rate by strain SSZ01was estimated by dividing the initial STX concentration spiked into the bacterial cultures by the number of days until the toxin was no longer detected.

HPLC analysis of SXT and degradation products

SXT and potential degradation products were determined in bacterial culture supernatant using reverse phase HPLC with post-column derivatization and fluorescence detection according to the methods of Oshima (1995). A C8 column (150 mm × 4.6 mm) with a C8 guard cartridge was used for the analysis of all STX analogues (STXs, GTXs and C-toxins). The temperature for column oven was 40 °C for STX and 20 °C for GTXs and C-toxins. The flow rate was 0.8 mL/min with injection volume varying from 5 to 200 μL. The flow rate for post column reagents was 0.4 mL/min. The excitation and emission wavelengths were 330 nm and 390 nm, respectively. All samples were filtered through 0.45μm filters before analysis. Specifically, the mobile phase used for the analysis of STXs consisted of sodium heptane sulfonate (2 mM), diammonium hydrogen orthophosphate (25 mM, respectively) and 5% acetonitrile (pH 7.1 with phosphoric acid). For GTXs analysis, the mobile phase consisted of sodium heptane sulfonate (2 mM) and diammonium hydrogen orthophosphate (10 mM, pH 7.1 with phosphoric acid). The post column oxidant solution was 7 mM periodate in 50 mM diammonium hydrogen orthophosphate, adjusted to pH 7.8 with 5 M sodium hydroxide, and the post column acid solution was 0.5 M acetic acid. The oxidant solution was 7 mM periodate in 50 mM diammonium hydrogen orthophosphate (pH 7.8 with 5 M sodium hydroxide) and the acid solution was 0.5 M acetic acid. For the analysis of sulfocarbamoyl toxins C1 and C2, the mobile phase was tetrabutyl ammonium phosphate (1 mM, pH 6 with 1 M sodium hydroxide). The oxidant solution was 7 mM periodate in 50 mM potassium dihydrogen phosphate (pH9 with 5 M sodium hydroxide) and the acid solution was 0.5 M acetic acid. Toxin standards including gonyautoxin 2 and 3 (GTX2, GTX3), gonyautoxin 5 (GTX5), decarbamoylsaxitoxin (dcSTX), and N-sulfocarbamoyl toxins (C1, C2), were purchased through local company from the Institute for Marine Bioscience, National Research Council, Canada

Artemia salina toxicity assay

The Artemia salina assay was conducted to test the toxicity of parent STX (i.e., STX standard without bacteria) and STX degradation byproducts obtained during biodegradation experiments. Potential toxicity of STX degradation byproducts was carried out for bacterial cultures showing complete degradation of STX (i.e., not-detectable by ELISA). The assay was performed using freshly hatched A. salina L nauplii according to Kiviranta et al. (1991) and Tokodi et al. (2018). The mortality was estimated after 48h and expressed as the difference (%) between mortalities in the tested and control samples following this formula-—%mortality = [(Test−Control)/(1−Control)] × 100. The lethal concentration causing death of 50% (LC₅₀) with 95% confidence limits was calculated by the probit analysis method (Finney, 1971)

Statistical analysis

All experiments were performed in triplicate. Variations in STX biodegradation by strain SSZ01between different initial toxin concentrations, temperatures and pH levels were evaluated using one-way ANOVA (P< 0.05).

STX biodegradation

The growth curve of strain SSZ01 cultivated with different concentrations of STX (0.5- 50µg L^-1) is shown in figure 1. The bacterial strain showed no lag period either in STX-treated or control cultures. The growth of this bacterium increased in both control and STX-treated cultures (P> 0.05) until the day 3 of incubation period. Subsequently, the bacterial growth decreased sharply in control cultures, but increased in all STX-treated cultures with a decline phase obtained at day 6 (Fig. 1). The growth of this strain also varied significantly (F3.1 =110.9, P<0.0001) and increased with initial STX concentrations, with no remarkable difference between the highest two STX concentrations (20 & 50µg L^-1). In addition to its survival, Bacillus strain SSZ01 was also able to degrade STX with varying extent depending on initial toxin concentration (Fig. 2). STX concentrations showed a slight decrease during the first day of incubation in treated bacterial cultures. Thereafter, these concentrations declined gradually and they became undetectable by day 8 at most. At the same time, STX concentrations in control cultures (i.e., without bacteria) showed no remarkable change along the incubation period, confirming that STX degradation occurred by bacteria rather than abiotic factors. Additionally, non-viable heat-treated bacteria showed no significant decrease in STX concentration during all the experiment period (therefore data are not shown), indicating that STX removal does not occur through adsorption on bacterial surface. The decrease in STX concentrations by strain SSZ01 differed significantly (F3.1=111, P <0.0001) with the initial STX concentrations. Additionally, the STX biodegradation rate by strain SSZ01also increased (F3.1=318.7, p< 0.0001) with the increase in the initial STX concentration. The highest biodegradation rate (10µg STX L^-1 day^-1) was obtained at the highest initial STX concentration (50µg L^-1) (Fig.3A), while the lowest rate (0.06µg STX L^-1 day^-1) was recorded at the lowest initial STX concentration (0.5µg L^-1). Complete degradation of STX by strain SSZ01also depended on the initial toxin concentration, as it occurred more rapidly within 5 days at higher STX concentrations (10, 20 & 50µg L^-1), compared with 6, 7 and 8 days at lower concentrations (5, 1, and 0.5µg L^-1, respectively) (Fig.2).

Effect of Temperature and pH on STX biodegradation

Figure 3 shows the biodegradation curves of STX by strain SSZ01incubated at various temperatures (10, 20, 25 and 30℃). The STX degradation rate increased with increasing temperatures (F4.07= 179, P < 0.0001), with highest degradation ratios obtained at 25 and 30 ºC (83% and 1005, respectively) (Fig.3). STX was completely removed by this bacterium within 4 days at 30ºC, compared to 6 days at 25ºC. On the other hand, the degradation activity of strain SSZ01 decreased sharply at lower (10 & 20ºC) and the toxin was not completely removed until day 8, with degradation ratios of 29% and 75%, respectively (Fig.3). In addition to temperature, the pH of growth cultures also affected significantly STX degradation by strain SSZ01 (Fig 4). The degradation ratio varied significantly (F4.07= 55, P < 0.0001) between the pHs tested during the present study (Fig.5). After 5 days of incubation, complete degradation of STX (i.e., 100% degradation ratio) was observed at pH8. This ratio decreased at lower pHs (6,7) (19 and 62%, respectively) and higher pHs (9,10) (88 and 58%, respectively) (Fig.4). Similarly, the STX degradation rate differed markedly with the pH of culture medium. The highest degradation rate was obtained at pH8 (10 µg L^-1 d^-1), while it decreased sharply at slightly acidic - pH6 (2.4 µg L^-1 d^-1), neutral-pH7 (6.3µg L^-1 d^-1) and alkaline conditions -pH9 and pH10 (7.1µg L^-1 d^-1 and 6.3µg L^-1 d^-1, respectively) (Fig. 5).

Potential biodegradation byproducts

Fig.6 shows the HPLC analysis of bacterial supernatants of strain SSZ01incubated with STX of concentration of 50 µg L^-1 at 72 and 120 h. HPLC chromatogram of control samples containing bacterial cells incubated in the absence of STX did not show any peak (Fig.7a). HPLC chromatograms showed that the retention time for STX was 6.3 min (Fig. 6b). An unknown peak was observed at retention time of 2.8 min as a probable degradation product of STX by strain SSZ01. This peak increased with the incubation period, in concomitance with the decrease in the peak of the parent SXT (Fig.6 b,c). After 120 h of incubation (i.e., the time of complete degradation of STX), the SXT peak vanished totally with increase in the unknown peak (Fig.6d). Compared to HPLC chromatograms of SXT analogues used as standards in our study (Fig. 7a,b), the unknown peak (i.e., degradation byproduct peak) was corresponding to C1 (Fig. 7c). With continued incubation, no other change in byproduct profile was seen (data not shown).

Toxicity of STX and SXT degradation byproducts

Table 1 shows the mortality of A. salina caused by parental STX and its degradation byproducts obtained during degradation experiments. The parent STX exhibited severe mortality to Artemia, and this mortality increased with the increase of STX concentrations with an LC₅₀value of 9.4µg L^-1. On the other hand, bacterial cultures showing complete STX degradation showed no toxicity to Artemia.

The present study provides evidence of bacterial biodegradation of STX by a Bacillus strain, isolated from eutrophic lake. To our knowledge, STX biodegradation by a typical strain of bacteria has never been reported before. However, STX degradation demonstrated by previous studies was either attribute d to a mixed bacterial population found in the bioactive filtration beds of water treatment plant (Kayal et al., 2008) or to unidentified marine bacteria isolated from the digestive tract of blue mussels (Donovan et al., 2008). Therefore, our study is the first to recognize and identify a bacterial strain (Bacillus flexus SSZ01) as STX-degrader. Strain SSZ01 grew well and showed no lag phase in the presence of STX in bacterial cultures. The absence of lag phase was also observed during CYN biodegradation by this strain (Mohamed et al. 2022). These results support the hypothesis that biodegradation of cyanotoxins takes place rapidly by bacterial strains with a previous history of these toxins in the aquatic environment (Smith et al. 2008; Mohamed and Alamri 2012; Mohamed et al. 2022). By co-existing with toxic cyanobacterial blooms in the same environment, our strain could have been frequently exposed to this toxin produced by cyanobacteria. Additionally, strain SSZ01 went into the decline phase earlier (after 3 days) in control cultures (i.e., without STX) compared to 5 days in STX-treated cultures. This suggests that this bacterium could use STX as a source of carbon and nitrogen for its growth, after exhaustion of the natural organic matter found in lake water used in our batch experiments. Similarly, earlier studies have shown that Bacillus strains can grow in sediments and lake water supplemented with other cyanotoxin, cylindrospermopsin, but without any addition of natural organic matter (Mohamed and Alamri 2012; Mohamed et al. 2022). It is interesting to note that, strain SSZ01 showed a more rapid complete degradation of STX at higher initial concentrations (10, 20 and 50 µg L^-1) than at lower concentrations (0.5-5 µg L^-1). This reflects that STX would act as an inducer activating the genes expressing the enzymes involved in the toxin degradation. Similar bacterial biodegradation results were also reported for another cyanotoxin (CYN) by strain SSZ01 (Mohamed et al. 2022), and by different bacterial strains (Smith et al. 2008; Mohamed and Alamri 2012), where complete degradation occurred at higher initial toxin concentrations but not observed at lower levels.

This apparently the most common feature of the biodegradation of many organic pollutants. It has been found that genes involved in the biodegradation of aniline by Pseudomonas sp. have been induced by aniline (Thomas and Peretti 1998), and the biphenyl degradation genes have been induced by biphenyl (Ohtsubo et al. 2000).

In our study, the regression analysis revealed a positive correlation (R²=0.97) between STX biodegradation rate and its initial concentrations. The highest rate (10 µg L^-1 day^-1) was achieved at highest initial STX concentrations (50 µg L^-1), and this rate declined markedly (0.06-0.83 µg L-1 day^-1) at lower initial toxin concentrations (0.5-5µg L^-1). This finding is in agreement with that obtained by Donovan et al. (2008) demonstrating that complete degradation of STX and neo-STX by unidentified marine bacteria strains occurred within 1-3 days, with a rate following first-order kinetics. Our results therefore confirm the suggestion that cyanotoxin biodegradation by bacterial populations is initial-concentration-dependent (Smith et al. 2008; Alamri 2010; Mohamed and Alamri 2012). This may be due to the possible existence of a threshold level of STX required to induce STX degrading genes in this bacterium.

Our study also found that STX degradation rate by strain SSZ01 was temperature-dependent. The highest degradation rate (12.5µg L^-1 day^-1) was achieved at the highest temperature (30ºC), and the temperature decline below 25ºC slowed this rate by a factor of 1.5-8. Such a high optimum temperature for STX degradation by strain SSZ01 is rational, since this strain has been isolated from a warm lake. Similarly, temperatures between 25 to 35 ºC have been shown to be favorable for the biodegradation of other cyanotoxins including microcystin (Ho et al., 2007, 2010) and CYN (Smith et al. 2008; Klitzke and Fastner 2012; Mohamed and Alamri 2012). These studies suggested that low temperature may negatively affect the biodegradation rate of cyantoxins by delaying the expression of cyanotoxin degrading genes, decreasing the activity of the enzymes involved in cyanotoxins degradation and/or decreasing the rate of cyanotoxin diffusion into the cell. In addition to temperature, pH also influenced the biodegradation rate of STX by strain SSZ01. The highest degradation rate was recorded at pH8 (10 µg L^-1 day^-1), and this rate decreased significantly under acidic, neutral and alkaline conditions. This reflects that STX degradation by this bacterial strain has an optimum pH for the activity of its degradation enzyme. Similar effects of pH on toxin biodegradation were also demonstrated for CYN by another Bacillus strain (AMRI-03) (Mohamed and Alamri 2012), with highest biodegradation rate obtained at pH7 and 8, and lowest recorded at higher pH levels (pH10 and 11). Also, Smith and Sutton (1993) showed that ATX-a biodegradation by sediment microbial populations occurred more rapidly at neutral pH (5 days), compared to 14 days at pH8 and 10, and 21 days at pH4. Although STX did not undergo a complete degradation by strain SSZ01at higher pH levels in our study, this bacterium nevertheless makes a contribution to STX degradation under alkaline conditions, in particular at pH9 and 10. The biodegradation of STX at pH9 and 10 might have important implications in water sources with heavy cyanobacterial blooms, which increase water column pH to alkaline levels (pH9-11) (Zepernick et al. 2021).

In our study, strain SSZ01 was found to transform SXT to C1 toxin, which was verified using an authentic standard by HPLC. C1 toxin is doubly-sulphated sulfocarbamoyl derivative of SXT and is considered the least toxic variant of SXT analogues (Donovan e al. 2008; Ho et al.2012; Lukowski et al. 2019; Raposo et al. 2020). C1 toxin is 165 times less toxic than SXT (Wiese et al., 2010). It seems that C1 toxin is the only product generated from SXT degradation by strain SSZ01, where no other degradation products of STX were identified. Previous studies reported the capability of some bacteria to sulfate STX analogues to less toxic sulfocarbamoyl derivatives via sulfotransferases (Sako et al., 2001). The generation of the disulfated C-toxins from STX by the sulfotransferases was also demonstrated cyanobacteria (Lukowski et al.2019)

The toxicity as estimated by A. salina assay of this byproduct was eliminated, indicating that the product of bacterial degradation of STX by strain SSZ01 is benign. A. salina assay proved to be suitable method to gives a value of the total acute toxicity of many cyanotoxins including microcystin (Kiviranta et al., 1991; Tokodi et al. 2018), cylindrospermopsin (Metcalf et al., 2002), anatoxin-a (Lahti et al., 1995) and saxitoxin (Mohamed and Al-Shehri 2015; Tokodi et al. 2018). Recently, the disulfated C-toxins derived from STX by sulfotransferases

have greater potential as safe pharmaceutical agents for the treatment of numerous neurodegenerative diseases due to voltage-gated sodium channels (VGSCs) disorders (Lukowski et al.2019).

The present study clearly demonstrated the ability of the bacterial strain SSZ01 to degrade STX to disulfated sulfocarbamoyl derivative (C1). STX biodegradation rate increased with the increase of initial STX concentrations. The study also revealed that STX biodegradation by strain SSZ01 was greatly influenced by temperature, with highest biodegradation rate obtained at 30 ºC. Moreover, pH of the cultures played a significant role in STX biodegradation by this strain. The highest STX degradation rate was achieved at pH8, and this rate underwent significant decrease at lower and higher pH values. The biodegradation of STX by bacteria under alkaline conditions (pH9,10) is of important implications in water sources containing heavy cyanobacterial blooms causing pH to rise to alkaline levels. Based on A. salina toxicity assay, our study also showed that the byproduct of STX degradation (C1) by strain SSZ01 was nontoxic. Therefore, this bacterium could be applied in slow sand filter to remove STXs in drinking water treatment plants. However, this necessitates setting up microcosm or mesocosm experiments to study the potential effects of this bacterial strain on water quality. Furthermore, such a nontoxic degradation product of SXT would be assayed in a further study for its potential therapeutic effects towards VGSC disorders.

Ethical Approval and Consent to Participate: Not applicable

Consent to Publish: Not applicable

Authors Contributions: MH, SA and YM cultured and determined the growth of Bacillus strain. ZM designed STX degradation experiments, and was a major contributor in writing the manuscript. All authors took part in data analysis and interpretation, read and approved the final manuscript.

Funding: The authors extend their appreciation to the Deanship of Scientific Research, King Khalid University for funding this work through research groups program under grant number R.G.P. 1/97/43, was used in the design experiments and collection, statistical analysis, data interpretation and writing the manuscript.

Availability of data and materials: Not applicable

Acknowledgements: The authors extend their appreciation to the Deanship of Scientific Research, King Khalid University for funding this work through research groups program under grant number R.G.P. 1/97/43.

Conflict of interest: The authors declare that there is no conflict of interest.

Alamri SA (2010) Biodegradation of microcystin by a new Bacillus sp. isolated from a Saudi freshwater lake. Afr J Biotechnol 9:6552–6559. http://www.academicjournals.org/AJB
Alamri SA (2012) Biodegradation of microcystin-RR by Bacillus flexus isolated from a Saudi freshwater lake. Saud J Biol Sci 19:435–440. https://doi.org/10.1016/j.sjbs.2012.06.006
Burford MA, Beardall J, Willis A, Orr PT, Magalhaes VF, Rangel LM, Azevedo SMFOE, Neilan BA (2016) Understanding the winning strategies used by the bloom-forming cyanobacterium Cylindrospermopsis raciborskii. Harmful Algae 54:44–53. Available from: http://dx.doi.org/10.1016/j.hal.2015.10.012 PMID: 28073481
Christoporidis C, Zervou S-K, Manolidi K, Katsiapi M, Moustaka-Gouni M, Kaloudis T, Triantis TM, Hiskia A (2018) Occurrence and diversity of cyanotoxins in Greek lakes. Sci Rep 8:17877. https://doi.org/10.1038/s41598-018-35428-x
Donovan CJ, Ku JC, Quilliam MA, Gill TA (2008) Bacterial degradation of paralytic shellfish toxins. Toxicon 52:91–100. https://doi.org/10.1016/j.toxicon.2008.05.005
Etheridge SM (2010) Paralytic shellfish poisoning: Seafood safety and human health perspectives. Toxicon 56:108–122. https://doi.org/10.1016/j.toxicon.2009.12.013
Finney D (1971) Probit analysis, third edn. Cambridge University Press, Cambridge
Palazot S (2010) Investigations into the biodegradation of microcystin-LR in wastewaters. J Hazard Mater 180:628–633. https://doi.org/10.1016/j.jhazmat.2010.04.081. Sawade E, Newcombe G, Saint CP, Brookes JD ()
Ho L, Hoefel D, Saint CP, Newcombe G (2007) Isolation and identification of a novel microcystin-degrading bacterium from a biological sand filter. Water Res 41:4685–4695. https://doi.org/10.1016/j.watres.2007.06.057
Ho L, Tang T, Monis PT, Hoefel D (2012) Biodegradation of multiple cyanobacterial metabolites in drinking water supplies. Chemosphere 87:1149–1154. https://doi.org/10.1016/j.chemosphere.2012.02.020
Jebeli MA, Maleki A, Amoozegar MA, Kalantar E, Izanloo H, Gharibi F (2017) Bacillus flexus strain As-12, a new arsenic transformer bacterium isolated from contaminated water resources. Chemosphere 169:636–641. https://doi.org/10.1016/j.chemosphere.2016.11.129
Kamp L, Church JL, Carpino J, Faltin-Mara E, Rubio F (2016) The effects of water sample treatment, preparation, and storage prior to cyanotoxin analysis for cylindrospermopsin, microcystin and saxitoxin. Chem Biol Interact 246:45–51. https://doi.org/10.1016/j.cbi.2015.12.016
Kayal N, Newcombe G, Ho L (2008) Investigating the fate of saxitoxins in biologically active water treatment plant filters. Environ Toxicol 23:751–755. https://doi.org/10.1002/tox.20384
Kiviranta J, Sivonen K, Lahti K, Luukkainen R, Niemela SI (1991) Production and biodegradation of cyanobacterial toxins – a laboratory study. Arch Fur Hydrobiol 121:281–294
Klitzke S, Fastner J (2012) Cylindrospermopsin degradation in sediments: The role of temperature, redox conditions, and dissolved organic Carbon. Water Res 46:1549–1555. https://doi.org/10.1016/j.watres.2011.12.014
Lahti K, Ahtiainen J, Rapala J, Sivonen K, Niemelä SI (1995) Assessment of rapid bioassay for detecting cyanobacterial toxicity. Lett Appl Microbiol 21:109–914. https://doi.org/10.1111/j.1472-765X.1995.tb01019.x
Lezcano M, Morón-López J, Agha R, López-Heras I, Nozal L, Quesada A, ElShehawy R (2016) Presence or absence of mlr genes and nutrient concentrations codetermine the microcystin biodegradation efficiency of a natural bacterial community. Toxins (Basel) 318:1–17. doi:10.3390/toxins8110318
Li J, Shimizu K, Akasako H, Lu Z, Akiyama S, Goto M, Utsumi M, Sugiura N (2015) Assessment of the factors contributing to the variations in microcystins biodegradability of the biofilms on a practical biological treatment facility. Bioresour Technol 175:463–472. doi:10.1016/j.biortech.2014.10.047
Li J, Persson KM (2021) Quick detection method for paralytic shellfish toxins (PSTs) monitoring in freshwater -A review. Chemosphere 265:128591. https://doi.org/10.1016/j.chemosphere.2020.128591
Lukowski AL, Denomme N, Hinze ME, Hall S, Isom LL, Narayan ARH (2019) Biocatalytic Detoxification of Paralytic Shellfish Toxins. ACS Chem Bio 14:941–948. https://doi.org/10.1021/acschembio.9b00123
Merel S, Walker D, Chicana R, Snyder S, Baurès E, Thomas O (2013) State of knowledge and concerns on cyanobacterial blooms and cyanotoxins. Environ Int 59:3030–3327. https://doi.org/10.1016/j.envint.2013.06.013
Metcalf JS, Beattie KA, Saker ML, Codd GA (2002) Effects of organic solvents on the high-performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction. FEMS Microbiol Lett 216:159–164. https://doi.org/10.1111/j.1574-6968.2002.tb11430.x
Mohamed ZA (2018) Potentially harmful microalgae and algal blooms in the Red Sea: Current knowledge and research needs. Mar Environ Res 140:234–242. https://doi.org/10.1016/j.marenvres.2018.06.019
Mohamed ZA, Al-Shehri AM (2015) Biodiversity and toxin production of cyanobacteria in mangrove swamps in the Red Sea off the southern coast of Saudi Arabia. Bot Mar 58:23–34. https://doi.org/10.1515/bot-2014-0055
Mohamed ZA, Alamri S, Hashem M (2022) Simultaneous biodegradation of harmful Cylindrospermopsis raciborskii and cylindrospermopsin toxin in batch culture by single Bacillus strain. Environ Sci Pollut Res 29:5153–5161. https://doi.org/10.1007/s11356-021-16062-z
Mohamed ZA, Alamri SA (2012) Biodegradation of cylindrospermopsin toxin by microcystin-degrading bacteria isolated from cyanobacterial blooms. Toxicon 60:1390–1395. https://doi.org/10.1016/j.toxicon.2012.10.004
Munoz M, Cirés S, de Pedro ZM, Colina J, Velásquez-Figueroa Y, Carmona-Jiménez J, Caro-Borrero A, Salazar A, Santa MaríaFuster MC, Contreras D, Perona E, Quesada A, Casas JA (2021) Overview of toxic cyanobacteria and cyanotoxins in Ibero-American freshwaters: challenges for risk management and opportunities for removal by advanced technologies. Sci Total Environ 761:143197. https://doi.org/10.1016/j.scitotenv.2020.143197
Nishu SD, Kang Y, Han I, Jung TY, Lee TK (2019) Nutritional status regulates algicidal activity of Aeromonas sp. L23 against cyanobacteria and green algae. PLoS ONE 14(3):e0213370. https://doi.org/10.1371/ journal.pone.0213370
OhtsuboY, Nagata Y, Kimbara K, Takagi M, Ohta A (2000) Expression of the bph genes involved in biphenyl/PCB degradation in Pseudomonas sp. KKS102 induced by the biphenyl degradation intermediate, 2-hydroxy-6-oxo-6-phenylhexa-2,4- dienoic acid. Gene 256:223–228. https://doi.org/10.1016/S0378-1119(00)00349-8
O’Neill K, Musgrave IF, Humpage A (2016) Low dose extended exposure to saxitoxin and its potential neurodevelopmental effects: a review. Environ Toxicol Pharmacol 48:7–16. https://doi.org/10.1016/j.etap.2016.09.020
Oshima Y (1995) Post-column derivatization liquid chromatographic method for paralytic shellfish toxins. J AOAC Int 78:528–532
Pereira P, Dias E, Franca S (2004) Persistence of paralytic shellfish toxins in freshwater environments. In: Steidinger KA, Landsberg JH, Tomas CR, Vargo GA (Eds), Proceedings of the Xth International Conference on Harmful Algae, October 2002. Florida Fish and Wildlife Conservation Commission, Florida Institute of Oceanography, and Intergovernmental Oceanographic Commission of UNESCO, pp.166–168
Raposo MIC, Gomes MTSR, Botelho MJ, Rudnitskaya A (2020) Paralytic Shellfish Toxins (PST)-Transforming Enzymes: A Review. Toxins 12:344. https://doi.org/10.3390/toxins12050344
Sako Y, Yoshida T, Uchida A, Arakawa O, Noguchi T, Ishida Y (2001) Purification and characterization of a sulfotransferase specific to N-21 of saxitoxin and gonyautoxin 2 + 3 from the toxic dinoflagellate Gymnodinium catenatum (Dinophyceae). J Phycol. https://doi.org/10.1046/j.1529-8817.2001.00119.x. .37:1044 – 1051
Smith C, Sutton A (1993) Persistence of Anatoxin-a in Reservoir Water. FWR Report No FR0427. Retrieved on Februray 25, 2015 from the World Wide Web.
Smith MJ, Shaw GR, Eaglesham GK, Ho L, Brookes JD (2008) Elucidating the factors influencing the biodegradation of cylindrospermopsin in drinking water sources. Environ Toxicol 23:413–421. https://doi.org/10.1002/tox.20356
Thomas SM, Peretti SW (1998) Continuous culture dynamics for aniline metabolism by Pseudomonas sp. CIT1. Biotechnol Bioeng 58(19980405):1–12. https://doi.org/10.1002/(SICI)1097-0290. ::AID-BIT1>3.0.CO;2-J
Tokodi N, Drobac D, Lazić G, Petrović T, Marinović Z, Lujić J, Malešević TP, Meriluoto J (2018) Svirčev Z
Screening of cyanobacterial cultures originating from different environments for cyanotoxicity and cyanotoxins,Toxicon154:1–6. https://doi.org/10.1016/j.toxicon.2018.09.001
Trainer VL, Hardy FJ (2015) Integrative monitoring of marine and freshwater harmful algae in Washington state for public health protection. Toxins (Basel) 7:1206–1234. https://doi.org/10.3390/toxins7041206
Wiese M, D'Agostino PM, Mihali TK, Moffitt MC, Neilan BA (2010) Neurotoxic alkaloids: saxitoxin and its analogs. Mar Drugs 8:2185–2211. https://doi.org/10.3390/md8072185
WHO (2020) Cyanobacterial toxins: saxitoxins. Background document for development of WHO Guidelines for drinking-water quality and Guidelines for safe recreational water environments. World Health Organization, Geneva, Switzerland (WHO/HEP/ECH/. WSH/2020.4)
Zepernick BN, Gann ER, Martin RM, Pound HL, Krausfeldt LE, Chaffin JD, Wilhelm SW (2021) Elevated pH Conditions Associated with Microcystis spp. Blooms Decrease Viability of the Cultured Diatom Fragilaria crotonensis and Natural Diatoms in Lake Erie. Front Microbiol 12:598736. https://doi.org/10.3389/fmicb.2021.598736

Table 1 Mortality (%) of brine shrimp larvae (Artemia salina) Exposed to saxitoxin and byproduct of saxitoxin degradation by Bacillus strain SSZ01

Saxitoxin concentrations (µg L^-1)	Saxitoxin standard	biodegradation product
Saxitoxin concentrations (µg L^-1)	Mortality (%)	Mortality (%)
Seawater + shrimps	0	0
Bacterial cultures without STX	-	0
0.5	9	0
1	26	0
5	57	0
10	77	0
20	92	0
50	100	0
LC₅₀ (µg L^-1)	9.4	-

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Biodegradation and detoxification of saxitoxin by Bacillus flexus in batch experiments

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