Arsenic uptake and metabolism by a marine cyanobacteria PCC7002

. Cyanobacteria Synechococcus PCC7002 is a typical marine bacterium that can tolerate salinity at around 20 ppt. In this study, cyanobacterium PCC7002 cells were incubated in a controlled environmental chamber for one week. Three different arsenic V (As V ) concentrations were added to the culture to observe the cyanobacterium’s capability to arsenic uptake. The arsenic uptake was then compared to the control media (without As V addition). The higher the arsenic concentration added to the media, the more Arsenic occurs in the cells. Cyanobacteria cells could take up arsenic from their media at the range of 60-90% of the total arsenic exposed to the media, which showed their high arsenic tolerance. Speciation analysis using tandem HPLC and ICPMS showed that As-sugar is the main arsenic species in the cells, while inorganic arsenic is the main arsenic species in the media. Further analysis for signal confirmation using LCMS showed that the transformation of inorganic As V into organic arsenic (As-sugar) confirmed the biotransformation of arsenic in the cyanobacteria cells. This result proves the vital role of cyanobacteria in arsenic cycling and biotransformation in marine environments.


Introduction
Arsenic is a naturally occurring metalloid in diverse environments such as soil, water, and rocks [1].The presence of Arsenic in marine environments added to the background condition of both geogenic and anthropogenic inputs to the ocean [2].Arsenic in the environment may come from both natural and anthropogenic sources.Sourced from natural and anthropogenic, arsenic delivered into the marine environment is dissolved in the water or deposited on the sea floor.As V predominantly exists in seawater as a deprotonated oxo anion H2AsO4 -, resembling the primary seawater nutrient phosphate, which also primarily exists as H2AsO4 - [3].Because of its similarity to the phosphate anion and the lack of structure to differentiate between both, inorganic arsenic is readily taken up by marine primary producers using the same transport system as diproton phosphate acid, with it then being metabolized as the essential nutrient, phosphate.
As photosynthetic organisms, cyanobacteria play an important role in primary production and nutrient cycling.In addition to their ecological importance, cyanobacteria have a unique ability to interact with Arsenic, transforming the inorganic Arsenic from the environment into a different and new form of molecule species [4].
Cyanobacteria have developed various mechanisms to tolerate elevated levels of arsenic toxicity.Besides enzymatic or protein modification in their metabolic system, cyanobacteria may also possess efflux pumps that actively remove Arsenic from the cell or sequester it in specialized cellular compartments [5].These transporters are responsible for the intake of nutrients from the environment, including As V , that has similar to the essential nutrient.
Cyanobacteria have been widely used in experiments to unravel arsenic metabolisms and their fate in the marine environment because they are easier to take care of in the lab environment, and their genetics have been fully sequenced.There are several research related to the use of microalgae to understand the arsenic transformation in these small aquatic primary producers [1,[5][6][7], however, more data regarding the use of marine cyanobacteria is needed.Furthermore, understanding the arsenic transformation in these small primary producers will be helpful in understanding arsenic transformation in marine food chains because many small animals, such as shrimp, sea urchins, and crab larvae, feed on diatoms and cyanobacteria.Therefore, this research has two aims.Firstly, to investigate the total arsenic uptake in marine cyanobacteria PCC7002.The second aim is to understand the arsenic transformation in cyanobacteria using speciation techniques, which can further improve the understanding of cyanobacteria's role in arsenic cycling in marine environments.

Culture media
The liquid BG11 media with slight modification was used for cyanobacteria culture.Commercial artificial salt for aquarium (20‰) combined with nutrients, e.g., metal mix, K2HPO4 (30.5 mg/ L solution), and NaNO3 (1.5 g/ L solution).All nutrients are pro-analysis grade and purchased from Sigma Aldrich and Merck.Media salinity was maintained at 20 ‰.All equipment, such as glassware, stirrer bars, and media, were sterilized using an autoclave (Atherton Tiger, Australia).The culture was placed in a growth cabinet with a controlled temperature of 19-21 C and a light intensity of around 4000 lux.The light was programmed for 12 h dark and 12 h light.Magnetic stirrers were used to maintain the culture rotation and to ensure the cyanobacteria obtained enough light for their photosynthesis.

Arsenic exposure experiment
Fresh cyanobacteria culture PCC7002 (obtained from Research School of Biology, ANU, Australia) was added into 2L Erlenmeyer flasks with the composition of 50 mL culture in 2 L media.The culture was placed in a temperature-controlled growth chamber (Eurotherm, Victoria, Australia) and was sampled daily for one month.Each sample was analyzed using a UV-visible spectrophotometer (Thermo Scientific) to check their growth rate.
The arsenic exposure experiment was conducted in one week of incubation, during the highest cell number in the culture.There are four arsenic treatments to the culture, i.e., control (without arsenic addition), low exposure (5 mg/L), medium exposure (10 mg/L), and high exposure (15 mg/L).After one week of incubation, the cultures were harvested, and then cells and media were separated using a centrifuge (Benchtop centrifuge 5804, Eppendorf).The cyanobacteria cell culture was then freeze-dried (Labconco, USA) while the water media was filtered using 0.45 µm filter size (Sartorius) and kept in -18 C cold storage for further analysis.

Samples preparation
Total arsenic standards were diluted from 1000 µg/ml stock arsenic pure standard solution (Perkin Elmer) to 100 g/L.Standards series from 0, 0.5, 1, 5, 10, 20, 40, 80, and 100 ug/L were diluted in 2% HNO 3 using inline dilution during the analysis.Arsenic species were prepared separately.Inorganic arsenic standards in the forms of As III and As V were prepared from NaAsO 2 and Na2HAsO4.7H2O,respectively (Sigma Aldrich, Australia).Methylated arsenic species were prepared from CH3AsNa2O3.6H2O and (CH3)2As(O)OH.7H2O for both monomethylarsonic acid (MA) and dimethylarsinic acid (DMA).Other organic arsenic species such as arsenobetaine, arsenocholine, trimethylarsonio-propionate, arsenosugars, and tertramethylarsonium ion were prepared in-house and diluted to desired concentration from 1000 µg/L stock solutions.Blank samples consisted of reagents prepared and digested in parallel with the samples to control for background noise from the reagents and to detect any potential contamination.
Total arsenic samples were prepared using microwave digestion following the methods of US EPA 3051 for seaweed digestion.Around 0.100 g of dried sample was weighed and transferred to a 7 ml Teflon polytetrafluoroacetate digestion vessel (A.I.scientific, Australia).Concentrated HNO3, 1 ml and 100 µl of peroxide (30%, Merck) were added to the vessel and left open for 2 hours until all bubbles disappeared.Digestion was carried out using an MDS-81D microwave oven (CEM, USA) with three steps, 2 min at 650 W, 2 min at 0 W, and 45 min at 450W.Upon complete digestion, vessels were cooled at room temperature (25 C) for approximately 1 hour.All samples were then transferred into 10 ml vials and diluted to 10 ml and further diluted inline during the analysis using ICPMS to obtain the acid concentration in the samples at or below 2%.Water-soluble arsenic species were extracted using the water bath method described in Kirby and Maher [8].

Samples analysis using Instrumentation
Total Arsenic was analysed using an ICPMS (NexIon, 300D, Perkin Elmer, USA) with a plasma power of 1300 W and argon gas flow of 0.9 L min-1.For quality control of analyses, calibration standards 20 µg/L or blank were alternated and run every ten samples.Internal standards 6Li, 45Sc, 71Ga, 130Te, 115In, and 209Bi were added in order to monitor the performance of mass accuracy during analysis due to acid effects or instrument drift [9].Certified reference material (CRM) DORM-3 (NRC, Canada) was used as a standard for arsenic-containing foodstuffs during the analysis.Total Arsenic was measured in the CRM to calculate the accuracy of the analysis.
Speciation was performed using a coupled instrument of HPLC-ICPMS (Perkin Elmer USA, data not shown in this article).Further analysis for peak confirmation using LCMS (Agilent, USA).The analysis condition using LCMS is specified in Table 1.

Growth curve
The growth rate of the cyanobacteria after one month of incubation is presented in Fig 1.
There was a rapid growth in the first week of the incubation, then slowly decreased from the 10 th to 20 th Finally, the cyanobacteria growth becomes steady after 20 days of incubation.This data suggests that the highest growth rate of the cyanobacteria was in their first week of incubation, therefore, this first week is the most suitable timeframe for the arsenic exposure experiment as their cells were at the highest rate.

As total analysis
The concentration of total arsenic in the cyanobacteria cells increased with the increase of arsenic concentration in the growth media (Fig 2).This figure shows that the more arsenic in the environment, the higher the chance of accumulation in the cyanobacteria cells.During exposure experiments with varying arsenic concentrations, it was observed that after one week of incubation, all media had low arsenic concentrations.This finding demonstrates the impressive capacity of cyanobacteria to absorb arsenic from their environment at a range of 60 to 90% of total arsenic in the culture media.Even at the lowest arsenic exposure level (5 µg/l), which is considered the arsenic background in a relatively pristine marine environment [2], the availability of arsenic and the ability of marine primary producers to absorb it from the environment led to an accumulation of arsenic in the algae cells.Electrostatic interaction, surface complexation, ion exchange, and precipitation of water surrounding the microalgae increase their ability to bioaccumulate metal and metalloids from their surrounding environment [10].
Cyanobacteria have an important role in arsenic cycling in marine environments.As a primary producer, cyanobacteria pass arsenic into the higher trophic level of organisms.The high total arsenic in marine fish is associated with the high concentration of arsenic from their diet, which, in this case, cyanobacteria as the primary producers.

Arsenic speciation
Understanding the toxicity of arsenic in marine organisms cannot be relied solely on total arsenic data itself.Further analysis using speciation techniques is highly required to determine whether the arsenic species is able to pose a hazard.Although arsenic is well known as a king of poison, not all arsenic species are toxic.Inorganic arsenic such as As III and As V is categorized as carcinogenic, and a few species, such as arsenobetaine, is nonhazardous to any living organisms [11].However, among those, there are a significant number of arsenic species whose toxicity still needs to be uncovered.Analysis using HPLC-ICPMS showed that arsenic species in the algae cells were mostly in the form of organic arsenic molecules, mainly in the ribose forms (As-sugars, data not shown here).
Further analysis using LCMS (Agilent, USA) showed that the main ion precursors are 363.4m/z and 431.5 m/z, which are phosphate As-sugar and sulfonate As-sugar, respectively (Fig 3).This finding is similar to previous result that PO 4 sugar and Sulfonate sugars are the primary arsenoriboside forms in diatom and unicellular algae [12].Other research also stated that arsenosugars are the main arsenic species in freshwater cyanobacteria [5].After the run of multiple reaction monitoring (MRM) for 7 minutes, all four sugar signals appeared; however, the signal for glycerol As-sugar was very low (Fig 4).It can be said that there was a massive transformation of arsenic during the exposure experiment from As V that added to the media into As-sugars, mainly in the form of PO 4 As-sugar, SO3 As-sugar.SO4 As-sugar and Gly As-sugar were presented as minor arsenic species.
There was no sign of inorganic arsenic in the cyanobacteria tissue, showing that the bacteria have a high capability in the detoxification of arsenic.The arsenic transformation from inorganic species such as arsenate (As V ) and arsenite (As III ) is part of the detoxification process in marine primary producers to reduce the toxicity of the element in the metabolic system of the algae [13].

Conclusion
In conclusion, marine cyanobacteria PCC7002 has a significant role in arsenic metabolism and transformation.Its capacity to absorb arsenic from their environment up to 90% showed the high bioavailability of arsenic in marine environments.Although arsenic is present in the toxic form of arsenate (AsV), cyanobacteria transform most of the poisonous substance into the organic form of arsenic sugars with lower toxicity than inorganic forms.The arsenic transformation process is part of the detoxification process in the marine producer's metabolic system.

Fig 1 .
Fig 1.Cyanobacteria growth rate during one month of incubation.The first week had the highest rate of the PCC7002 cells in the culture.

Fig 2 .
Fig 2. Arsenic concentration (µg/g) in the cyanobacteria, growth media, and certified reference material (CRM).Control is cell culture without the addition of Arsenic; I = culture plus five µg/l As, II = culture plus ten µg/l As, and III = culture + 15 µg/l As.

Fig 3 .
Fig 3. Scanning for precursor ions of m/z products at 255, 121, 105 and 91.Precursors at 363.4 and 431.5 are the most apparent ion products that belong to PO4 As-sugars and SO3 As-sugars, respectively.

Table 1 .
LCMS condition for arsenosugar analysis