Chromatic Toxicity Bioassay of Water through Bacterial Ferricyanide Reduction

Water quality assessment demands a precise anatomization of specimens that comply with acclaimed water purity standards. Today, the growing number of toxicants and their synergistic consequences make it necessary to develop general toxicity assays able to examine and determine water pollution. Contemporary general toxicity methods hinder specimen analysis due to their prolonged operation protocols. Also, the equipment involved is very expensive that not everyone can afford it. In an effort to resolve these drawbacks, a quick and cost effective toxicity bioassay based on chromatic changes related to bacterial ferricyanide reduction is introduced here. E.coli cells (Model Bacteria) were stably conned on four supports: Cellulose-based Paper Discs, Silica 60, Polystyrene, and Acrylic Beads, which remained useful for a long period at -20ºC. Copper was used as a model toxic agent to perform Bioassay Assessment. Chromatic changes related to bacterial ferricyanide reduction were determined by visual inspection. Cellulose Paper Discs, Polystyrene, and Acrylic beads showed good results and better viability, while Silica 60 proved itself as a weak support and resulted in poor viability.


Introduction
Waterborne and related diseases not only deteriorate the environment and cause pollution, but they are also the reason of increasing rates in morbidity and mortality. Though efforts are continuously being made to preserve water safety, waterborne outbreaks are still reported worldwide. Diseases related to water cause more than 3.4 million deaths each year (Berman, 2009).
Water is a "Universal Solvent," as it is able to dissolve almost everything (Anne Marie Helmenstine, 2019).
This makes water essential for the proper functioning of the Earth's ecosystem. All water-related problems are a major public health concern all around the world. The foremost source of water pollution are the industries. Industries use freshwater to carry the wastes from different sources into rivers, lakes, and oceans. Water pollution has a negative impact on all living creatures and consequently the food chain as well. It can negatively affect the daily use of water from drinking to bathing. To get the proper solution to all these water-related problems, the treatment of water is necessary. Therefore, for the proper analysis and control of water quality, the detection of the pollutants and toxic contaminants is a major step. To detect any type of contaminant, a wide variety of tools, as well as advanced analytical methods, are required. Specialized, appropriate, speci c, and powerful diagnostic tools have been developed to detect various contaminants in water (Ramírez-Castillo, 2015), as well as chemical analysis techniques are also of great importance in determining speci c substances, but most of these tools and the techniques are limited in analysing a complex sample that contains contaminants in a huge variety. Also, they are timeconsuming, require lengthy protocols, and rely on very expensive instrumentation.
In recent years, the progress of biosensor has unlocked an extraordinary perspective to the onsite, easy, and money-saving monitoring of water quality (Chaubey A, 2002). In order to analyse contaminants of water, the innovative idea of biosensors is appropriate and convenient. By electrochemical means, an immense proportion of enzymatic biosensors are operated. Towards the target analyte, high sensitivity is the bene t of Enzymatic Biosensor (Eggins, 2013). Nevertheless, due to enzyme deactivation, it also faces drawbacks like time consumption, expensive protocols of enzyme puri cation and immobilization; as well as less life span and deprived stability (Jaffrezic-Renault, 2011).
As compared to Enzymatic Biosensor, Microbial Biosensor is more bene cial as it has more simplicity associated with biocatalyst production, predominantly when huge quantities are needed. To a large variety of analytes, microbial biosensors are more exible and tactful (Jaffrezic-Renault, 2011). Mainly Microbial Biosensors have been explored as water quality monitoring devices, and presently few prototypes used as water toxicity sensors are also commercialized. Microbes that remain alive under high alkaline, acidic, high temperature, and saline conditions give ways to great perspectives on water monitoring for industrial process waste monitoring (D'souza, 2001).
E.coli has the honour of being the most extensively studied microbial organism due to its various important roles in elds related to biotechnology, industrial sciences, medicine, and biological sciences; and in recent years, it has gained quite a positive reputation in development of Microbial Biosensors.
Being a model organism for laboratories, almost every aspect of E.coli, from its genome to its proteinmaking capability, has been exploited (Lee, 2009). In bacterial metabolism, oxygen acts as the hydrogen acceptor in the TCA cycle under the aerobic condition for growth and respiration (Hadjipetrou, 1966).
However, under anaerobic conditions, an arti cial hydrogen acceptor can be utilized by the bacterial metabolism as well. This arti cial hydrogen acceptor can be a nitrate, sulphate, benzoquinone, or ferricyanide. We used ferricyanide as a hydrogen acceptor because after accepting the hydrogen, it gets reduced into ferrocyanide, and this whole reaction is accompanied by a change in colour (Pujol-Vila, 2015). Bacteria are termed immortal because they do not die a natural death, but they only die because of abiotic factors like temperature, pH, and insu cient nutrient supply (Bickerstaff, 1997).
Several assays indirectly assess the activity of microbial cells by quantitatively analysing changes to the bacterial metabolic system (Bu , 2011), speci cally to the electron transport chain. The electron transport chain is selected because it maintains the basic integrity of the cell. There are various aspects of analysing the microbial metabolism, and it can be analysed by measuring inhibition of respiration, by qualitatively assessing the accumulation of ferrocyanide in ferricyanide mediated bioassays or by quantifying bioluminescence. The incubation of bacteria that can utilize ferricyanide as an alternate terminal electron acceptor is referred to as ferricyanide-mediated respirometry assay (FM-RES assay) (Catterall, 2010). Our experiment revolves around the fact that in the presence of a heavy metal such as copper, lead, mercury, chromium, cadmium, etc., this metabolic conversion of ferricyanide into ferrocyanide altogether stops. The bacteria are unable to reduce ferricyanide; thus, no ferrocyanide is produced. We are using this principle to detect the presence of heavy metals in water.

Chemicals and Samples
Luria-Bertani Broth, MacConkey Agar, Potassium Ferricyanide, Copper Sulphate, Glucose, Potassium Dihydrogen Phosphate, Dipotassium Hydrogen Phosphate, Deionized Water, Silica 60 (high purity grade), 9mm Cellulose Paper Discs (0.7mm thickness), Acrylic Beads, and Polystyrene. All chemicals were of high systematic grade, and all solutions were prepared with distilled water under strictly sterile conditions. NaCl was used to rehydrate disc before streaking it on agar. Water samples were prepared with Copper Sulphate in 50mL Falcon tube at a ratio of 1:2 and 1:4. Concentrated CuSO4 was diluted with water in the ratio 1:4 (one part of concentrate is mixed with four parts of water) and 1:2 (one part of concentrate is mixed with two parts of water).

Preparation of E.Coli Culture and Bacterial Paper Discs (BPDs)
E.coli was grown aerobically in Luria-Bertani (LB) broth for 18 hours at 37ºC. The grown culture was centrifuged at 10100 x g for 15 minutes and then re-suspended in 0.1M phosphate buffer containing 2% glucose to a 2.0x1010 ± 0.5x1010 cell mL-1 bacterial concentration. By the adsorption method of immobilization, E.coli was entrapped in 9mm Cellulose Paper Discs (PDs) (0.7mm thickness), which were used as the supporting material. For E.coli cell entrapment on PDs, we inoculated one side of the disc with a bacterial suspension of volume 60 µl and dried it at room temperature (25 ºC) for 2 hours in Laminar Flow Hood (F. Pujol-Vila, 2016). After being completely dried, PDs were stored at -20ºC until further required.

Bacterial Viability Checking
For evaluating bacterial viability in the immobilized paper discs, the immobilized paper discs were rehydrated by putting them in 0.9% (w/v) NaCl then shaken with vortex for 5 minutes to re-suspend the immobilized bacteria. Bacterial viability was determined by plating the suspension on MacConkey agar and then they were incubated for 24 hours. Similar protocol was followed for evaluating bacterial viability in E.coli immobilized Silica 60, Polystyrene, and Acrylic Beads.

Immobilization of E.coli on Silica 60
For the E.coli entrapment, 20ml of E.coli culture (containing 1M phosphate buffer (PB) and 2% glucose) and 10ml of 50mM phosphate buffer (immobilization buffer) with pH 7.0 was added to 1g of silica 60. Silica 60 is the matrix for immobilization.
The immobilization was carried out in a 100 mL glass vessel placed on a magnetic stirrer at 200 rpm, for an hour, at 25ºC. Then it was dried at room temperature (25 ºC) for 24 hours in a Laminar Flow Hood and stored at -20ºC until further required (Sugahara, 2014).

Immobilization of E.coli on Polystyrene
For the immobilization of E.coli cells, Polystyrene was used as third support material, and we made small fragments of it prior to immobilization. Polystyrene was hydrated with E.coli culture (containing 1M phosphate buffer (PB) and 2% glucose). Then it was dried at room temperature (25 ºC), for 24 hours in a Laminar Flow Hood and stored at -20ºC until further required (Hackel, 1975

Immobilization of E.coli on Acrylic Beads
For the entrapment of E.coli cells in Acrylic Beads, we placed the beads in an aqueous buffer containing E.coli cells culture (containing 1M phosphate buffer (PB) and 2% glucose). The immobilization was carried out in a 50mL Falcon tube, by placing it in a Laminar Flow Hood for 12-72 hours, at room temperature. After being completely dried, Acrylic Beads were stored at -20ºC until further required (Boller, 2002). We then checked for a change in colour with a reaction mixture that contained 20µl of 1:2 diluted CuSO4 solution instead of 1:4 diluted CuSO4 solution, and after 24 hours, the reaction mixture produced no change in colour. This was inferred to the increased concentration of copper in the reaction mixture which hindered the conversion of ferricyanide into ferrocyanide.

Analytical performance of E.coli immobilized polystyrene in visual inspection of colour change
The E.coli immobilized polystyrene fragments were inoculated in 20µl of 1:4 diluted CuSO4 and 10µl of potassium ferricyanide solution. After 24 hours, the reaction mixture showed an obvious change in colour.
We then checked for a change in colour with a reaction mixture that contained 20µl of 1:2 diluted CuSO4 solution instead of 1:4 diluted CuSO4 solution, and after 24 hours, the reaction mixture produced no change in colour. This was also inferred to the increased concentration of copper in the reaction mixture that hindered the conversion of ferricyanide into ferrocyanide.
4.5 Bacterial cell entrapment and adsorption e ciency and stability on cellulose paper discs, silica 60, acrylic beads, and polystyrene The entrapment capacity of bacterial cells on the chosen solid hydrophobic matrices was seen to be different. We checked it by putting each E.coli immobilized solid matrix through a growth test in Nutrient Broth.
For cellulose paper discs, we checked the entrapment capacity through inoculation in 10ml nutrient broth containing test tubes and later on incubating them at 37ºC. We then checked for bacterial growth in the broth after 24 hours. The presence of E.coli pellicle in the broth con rmed the successful immobilization of E.coli on cellulose paper discs.
Entrapment capacity for silica 60 was also checked in the same way as for cellulose paper discs. After 24 hours of incubation of silica 60 in nutrient broth containing test tubes, we observed slight turbidity in the growth media, which con rmed that E.coli immobilization had occurred but at a much lower scale in contrast to the cellulose paper discs.
Likewise, E.coli immobilized acrylic beads and polystyrene inoculation in nutrient broth and their incubation at 37ºC for 24 hours resulted in signi cant turbidity of the growth media, con rming successful entrapment of E.coli cells on the matrices.
The highest entrapment capacity was observed with cellulose paper discs as compared to the other three matrices as it produced a characteristic E.coli growth pellicle in nutrient broth. 4.6. Validation of results using two different concentrations of CuSO4 solution as model toxic compound In order to validate our result, we used two different concentrations of CuSO4: 1:4 and 1:2. In the reaction mixture that contained 1:4 CuSO4, the conversion of ferricyanide into ferrocyanide occurred with a visible change in colour. Reaction mixtures containing 1:2 CuSO4 did not produce any change in colour, which meant that the conversion of ferricyanide into ferrocyanide did not take place. This can be validated by using different concentration ranges of other heavy metal compounds apart from copper.

Conclusion
In this research, E.coli cells (used as model bacteria) were con ned on cellulose paper discs, Polystyrene, Acrylic beads, and Silica 60; and chromatic changes corresponding to bacterial ferricyanide reduction reaction was used for toxicity diagnosis. E.coli cells were stably con ned on Cellulose paper discs and showed good viability, whereas Polystyrene and Acrylic beads also showed good viability in 1:4 Copper Sulphate solution and much better viability in 1:2 Copper Sulphate solution. Silica 60 immobilization capacity was less than other supports, so it showed poor viability in a 1:4 copper sulphate solution. After validation of results with different concentrations of CuSO4 solution and checking entrapment capacity of the supports in nutrient broth, we conclude that cellulose paper discs showed the best detection results compared to acrylic beads, polystyrene and silica 60. Silica 60's entrapment capacity was the least among other supports, that is why it showed poor heavy metal detection results hence the reason for being considered inferior as a Bacterial cell support matrix.

Consent for Publication
Not Applicable.

Availability of Data and Materials
Not applicable. Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

Competing Interests
The authors declare that they have no competing interests.