Comparison of a Biosensor and a Commercial UV-Visible Method for Measuring Hexavalent Chromium in Liquid Medium

The objective of the research was to contrast two methods for the quantication of hexavalent chromium. The rst method is the biosensor that from the gene transformation of the cells of Escherichia coli, was incorporated by electroporation the plasmid pTOP Blunt V2, synthesized with luxA genes that provides luminescence through the catalytic activity of the luciferase top and chr genes that give the bacteria resistance to chromium. The second method is the application of the UV-visible colorimetric technique. Chromium was analysed at different concentrations, from 0.05 mg l −1 (maximum allowable limit for human consumption); 0.1 mg l −1 ; 0.2 mg l −1 ; 0.4 mg l −1 ; 0.8 mg l −1 and 1 mg l −1 with 5 replicates, subsequent to this, the two methods of chromium analysis were applied in river samples, thus obtaining that the biosensor in concentrations of 2x10 6 CFU of E. coli, has a margin of error of 1.4%, a result derived from the coecient of determination of the absorbance of chromium, unlike the UV-visible method with the colorimetric equipment, which presented a reading error of 3.9%.


Introduction
One of the methodologies currently applied to determine and quantify contaminants present in water are chemical analyzes, which can reach the detection of parts per trillion (ppt); since they are considered the most sophisticated. These chemical analyzes require pretreatment to preserve the sample during transfer to the laboratory, to avoid altering the results. This occurs, for example, in gas chromatography and mass detection analyzes; expensive and large equipment that requires its use in the laboratory (Schoffer et al., 2011).
Another type of process used corresponds to biological tests or bioassays; Some of the biosensors are devices composed of bacteria, combining biotechnology and microelectronics to form an analytical device, reducing the time used for the analysis of contaminants in the water. In this way, it is desired to implement new processes for the detection of toxic pollutants, e ciently replacing conventional methods at lower cost and time (Reyes, 2016).
The World Health Organization (WHO) considers chromium as one of the 10 most toxic and harmful transition metals for health and the environment, in its hexavalent form; currently exceeding the maximum permissible limits of 0.05 mg l −1 for water for human consumption (WHO, 2018).
The National Water Quality Strategy (ENCA) indicates that in Ecuador the quality of water has deteriorated in recent years due to anthropic activity and industrial chemical processes, producing waste with toxic organic and inorganic pollutant loads, such as chromium +3 and chromium +6 which are di cult to remove and together with the poor application of environmental regulations, progressive damage to the environment is being generated (ENCA, 2016). At the regional level, the de cient public service, the lack of control of the emission of ecological ow, indirectly cause the discharge of uncontrolled e uents into the environment by industrial and agricultural activities, generating mixed-type waste due to the lack of wastewater collection systems. This does not allow the correct separation of industrial and urban e uents, generating poor management of water resources, loss of biodiversity, loss of environmental services and deterioration of ecosystems (Llerena and Aguay, 2018).
The objective of the research was to compare two methods for the quanti cation of hexavalent chromium, these being a eld method known as colorimetry and the use of a bacterial biosensor that was manufactured for this study. This research has been developed to broaden the eld of study, regarding the methodologies used for the detection of hexavalent chromium in water sources, whether they are for human consumption, irrigation or water trough; thus guaranteeing the protection of the environment, being a shared responsibility between society and the government, that is, reducing the impacts of waste, inputs and processes; with the aim of achieving adequate environmental management in small, medium and large industries (Ortiz and Carmona, 2015).

Materials And Methods
This research was carried out in the Biotechnology Laboratory of the School of Agricultural and Environmental Sciences of the Ponti cia Universidad Católica del Ecuador Sede Ibarra.

Elaboration of the biosensor
For the elaboration of the biosensor, the sequence of the chr gene that gives the bacterium resistance to chromium and the sequence of the lux operon to generate the optical response of the biosensor was searched in the registry of community collection of genetic sequences available in iGEM (International Genetically Engineered Machine) to later assemble the plasmid in the Biotechnology company "Humanazing Genomics © MAROGEN Inc.". The result being the genetic synthesis described in Table 1 (MACROGEN Inc, 2018).
The result of the assembly of the gene sequences produced a plasmid with a size of 1,847 base pairs (bp) and a guanine and cytosine (GC) percentage of 60.23%.
For the modi cation of the genes of the E. coli bacteria, the Electroporation method described by Bastida and Levi (2019) was used, where a Thermo Scienti c ULT1230A freezer was used to prepare the competent cells, since these must be preserved at a temperature of 4°C. After this, 10 µl of the manufactured plasmid plus 20 µl of the competent cells were taken and added in a 2 ml eppendorf tube, this mixture was centrifuged and placed in the freezer again.
For the transformation of E. coli, the Electroporation Neon Device equipment was set at 2500 volts for 4 milliseconds and two pulses for the transmutation of E. coli (Gómez and Garns, 2003). To prepare the Electroporation equipment, the Neon Pipette Station was connected in which the Neon Tube cuvette was inserted with 3 ml of electrolytic buffer (Electrolytic Buffer) with a temperature of 2°C to 4°C (Gómez and Garns, 2003). LB Broth Base broth was prepared in a 250 ml boeco bottle, according to the manufacturer's instructions.
Next, 10 ml of this mixture was dispensed into 24 test tubes and sterilized with the help of the ICAN CLAVE autoclave for 40 minutes. Having the 24 test tubes already sterilized, hexavalent chromium concentrations of 0.05 mg l −1 were placed; 0.1 mg l −1 ; 0.2 mg l −1 ; 0.4 mg l −1 ; 0.8 mg l −1 and 1.0 mg l −1 .
Next, 100 µl, equivalent to 2x10 6 CFU of the transformed cells, was added to each test tube, incubated for 2 hours and chromium analysis was carried out by the EPOCH Microplate Spectrophotometer method, where 100 µl was added to the cells. samples that were previously incubated, placing the different chromium concentrations in each column of the microplate and 5 repetitions in each row. With the help of the EPOCH Microplate Spectrophotometer from Biotec Instruments and a computer program for data analysis Gen5 Software, the absorbance analysis of the samples was carried out to obtain the calibration curve (Macías et al., 2019).
Analysis of the process for the detection of chromium using a modi ed E. coli strain.
The E. coli bacteria were seeded in quantities of 2x10 6 CFU genetically transformed in test tubes with LB Broth broth from the manufacturer LAB a Neogen Company (Luria Bertani) ®, a medium that does not intervene in the reaction between the bacteria and the metal and provides the necessary nutrients for the bacteria, plus the transition metal, verifying their resistance and the optical response of the luciferase gene and the interaction between the metal and the medium (García et al., 2005). In the research carried out by Huelves (2008), the enzyme that catalyzes the luminescence reaction is encoded in the enzymatic protein of luciferase, an operon that was modi ed together with the chromium resistance genes by inserting into the E. coli bacteria, thus obtaining a medium that translates into light emission.
Field application of the two methods to quantify hexavalent chromium For the eld application of the two methods used to quantify hexavalent chromium, the research of Jurado et al. (2017). The rst method suggested uniformly sectioning the total length of the river, which consisted of 12.71 km, identifying accesses, the presence of contamination by wastewater discharges ( Figure 2) and agricultural activities, a method applied to date in the monitoring of river samples. Two levels were raised; the macro location that determined the most representative sections according to the total length of the Pichaví river; the micro-location that implied the equivalent division of the total length of the river, with a distance of 2 km between each point (Figure 3), a distance that must be considered according to the different methods applied in the eld for the collection of a sample, due to the estimation of the total uniform mixture of water between each section, thus generating a total of 6 points for the collection of water samples (Jurado and Mercado, 2017).
In the research, a statistical design of paired plots was applied by applying the t-Student test, in order to see the relationship between the methods applied for the measurement of hexavalent chromium and de ne the hypothesis. For the normality of the data, the Shapiro -Wilk test (Rossiter, 2014) was used. Two tests were used being these cases: the rst test used 6 concentrations of hexavalent chromium of 0.05 mg l −1 ; 0.1 mg l −1 ; 0.2 mg l −1 ; 0.4 mg l −1 ; 0.8 mg l −1 and 1.0 mg l −1 ; This served to standardize the measurement method and verify that bacteria have the potential to measure chromium. Subsequently, 6 liquid samples were taken from the Pichaví river and the biosensor technique was compared with a commercial and eld technique.
To compare the e ciency of the two methods, standard liquid samples and river samples were analyzed by the EPOCH Microplate Spectrophotometer from Biotec Instruments and with the SMART 2 Colorimeter from LaMotte Company, comparing the normality of the results by means of a Shapiro test. -Wilk.

Results
Analysis of the physical-chemical parameters of the river According to the results obtained in Table 2, the conductivity parameter of the 6 river water samples is within the permissible limits of the TULSMA; Total dissolved solids with a minimum value of 475 mg l −1 and a maximum of 516 mg l −1 of the 6 water samples, indicate values within the permissible ones. The results of the pH and BOD parameters indicate that the state of water quality in the environment is optimal for the development of organic matter and microorganisms, essential for oxygenation.
Evaluation of the biosensor with standard liquid samples and samples from the Pichaví river contaminated with the transition metal.
Biosensor applied to standard liquid samples Figure 4 shows the absorbance curve of the equipment, with a margin of error of 1.6% when quantifying a sample. The coe cient of determination, also known as R 2 , has a value of 0.9841, which is very close to 1 and through this result it is known that it is a reliable method to quantify hexavalent chromium.
To know the normality of the data obtained from the different chromium concentrations submitted by the biosensor, the Shapiro-Wilk test was applied, indicating the normal distribution of the data.
The result obtained in Table 3 represents the P value in the normality of the data. In the Shapiro-Wilk test, with a P-value of 0.11, it was obtained that in the values of the amount of chromium absorbance at different concentrations there is a normality of distribution.
Biosensor applied to river samples To know the normality and homogeneity of the data obtained from the different samples taken from the river submitted by the biosensor, the Shapiro-Wilk and Levene test was applied, indicating the normal distribution of the data and their homogeneity.
The data obtained in Table 4 represent the P value, both for normality and homogeneity. In the Shapiro-Wilk test, with a P-value of 0.95, it was obtained that in the data of the amount of Chromium absorbance there is normality.
On the other hand, it is mentioned that all the data have homogeneity of variance in their distribution since the P-value = 0.35 of the F is greater than the 0.05 level of signi cance, thus accepting the null hypothesis.
Comparison of the e ciency of the biosensor by the EPOCH Microplate Spectrophotometer method (BIOSENSOR) vs the UV-visible technique (Colorimeter).

EPOCH Microplate Spectrophotometer (BIOSENSOR) vs UVvisible technique (Colorimeter) at known chromium concentrations
For the comparison of the two methods applied to know their e ciency through a quantitative measurement at known chromium concentrations, it is distributed with 2 degrees of freedom, providing the hypothesis of equality of the means.
According to the results obtained, the statistical value calculated for the p-value of the t-Student test is 0.02; less than the signi cance level of 0.05, in the concentration of 0.05 mg l −1 of hexavalent chromium, in this way the null hypothesis is rejected.
According to the results obtained, the calculated statistical value of the p-value of the t-Student test is 0.00024; less than the signi cance level of 0.05, in the concentration of 0.1 mg l −1 of hexavalent chromium, in this way the null hypothesis is rejected.
According to the results obtained, the calculated statistical value of the p-value of the t-Student test is 0.07; greater than the signi cance level of 0.05; in this way the null hypothesis is accepted.
According to the results obtained, the calculated statistical value of the p-value of the t-Student test is 0.07; greater than the signi cance level of 0.05; in this way the null hypothesis is accepted.
According to the results obtained, the calculated statistical value of the p-value of the t-Student test is 0.002; less than the signi cance level of 0.05; in this way the null hypothesis is rejected.
According to the results obtained, the statistical value calculated for the p-value of the t-Student test is 0.02; less than the signi cance level of 0.05; in this way the null hypothesis is rejected.
According to the statistical analysis of the t-Student test applied for the quanti cation of hexavalent chromium by applying the EPOCH Microplate Spectrophotometer method (biosensor) and the UV-visible method by Colorimetry, the null hypothesis is rejected, since, the 6 different concentrations of chromium quanti ed, 4 of the concentrations indicate that the EPOCH Microplate Spectrophotometry method is different from the UV-visible method by colorimetry, accepting the alternative hypothesis.

EPOCH Microplate Spectrophotometer (BIOSENSOR) vs UVvisible technique (Colorimeter) applied in the eld
For the analysis of river samples, the two methods were used. EPOCH Microplate Spectrophotometry is different from the UV-visible method by colorimetry, applying a third method (Table 11); Atomic Absorption Spectroscopy, method suggested by (Peraza, 2011) for its precision, linearity of detection and quanti cation of metals.

Analysis of the physical-chemical parameters of the river
The results of the following parameters analyzed from the samples collected from the Pichaví River indicated in Table 2 (do not include  As Chaparro (2020) mentions, the determination of some physical-chemical parameters indicate the quality of water present in the environment to be analyzed, such as pH and BOD that are responsible for self-puri cation, causing the elimination of toxic polluting residues.

EPOCH Microplate Spectrophotometer (BIOSENSOR) vs UVvisible technique (Colorimeter) applied in the eld
To determine the e ciency of the methods applied to quantify chromium, the results of the different samples taken from the river were compared; For sample 2 it was obtained that the biosensor quanti ed 0.19 mg l −1 of chromium with a difference of 0.01 mg l −1 in relation to the analysis performed by Atomic Absorption.
According to Álvarez et al. (2013), the atomic absorption spectroscopy method is a validated test with reliable results when quantifying metals, being the most favorable due to the curve that is calibrated from a standard that passes through a ame and is crossed by a beam of light that contains speci c waves of the analyte, in this case hexavalent chromium, with precision and accuracy results, recommended for the comparison of quanti ed results with other methods.
When comparing the colorimetric method with the Atomic Absorption method of the result of sample two, a difference of 0.08 mg l −1 is obtained, and with a difference of 0.07 mg l −1 with the EPOCH method (BIOSENSOR).
Corroborating with the results obtained from the t-Student test, where the null hypothesis is rejected in most of the chromium concentrations quanti ed, where the EPOCH Microplate Spectrophotometry method (biosensor) and the UV-visible method by Colorimetry are totally different when measuring hexavalent chromium due to the comparison between the different results obtained by the applied methods.

Conclusions
Bacterial biosensors are an alternative as qualitative and quantitative analytical devices for metals, due to the reaction between the bacteria and the metal producing luminescence, which indicates the presence of the toxic agent analyzed thanks to the lux operon that was inserted into the bacteria and the resistance to chromium that was given by the chr genes.
The analyzes carried out with the bacterial biosensor show that it has a high speci city regarding the measurement of hexavalent chromium, in concentrations of 0.05 mg l −1 to 1.0 mg l −1 with an error of 1.6%, thus ensuring that the elaborated biosensor is effective in terms of metal quanti cation, accepting the hypothesis raised in the research.
The e ciency of the biosensor before the quantitative analysis in comparison with the UV-visible method is more e cient, because the UV-visible method uses a eld equipment, which presents a margin of error caused by the systematic factors in the processes of sampling, forcing the application of control samples, increasing residual sample. Bibliographic 1. Álvarez, C., Acevedo, R., & Severiche, C. (2013). Evaluación analítica para la determinación de aluminio, bario y cromo en aguas, por espectroscopia de absorción atómica con llama óxido nitroso-      Source: Own elaboration.  Source: Own elaboration. Figure 1 Quali cation of the biosensor at different chromium concentrations Source: Own elaboration.

Figure 2
Urban wastewater discharge point to the Pichaví river Source: Own elaboration.

Figure 3
Location of the water sampling of the Pichaví river Source: Own elaboration.

Figure 4
Absorbance of the EPOCH Microplate Spectrophotometer (BIOSENSOR) Source: Own elaboration.

Supplementary Files
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