Electrochemical Biosensor Using Acetobacter Enzyme for Detecting Alcohol

Determining halal product of fermented foods and bevearages becomes important for muslim consumers due to religious codification of halal. In order to help on site analysis, an alcohol biosensor was under development. The biosensor was constructed using bacteria producing alcohol oxidase (AOX). Bacteria from apple juice was isolated in advance to obtain pure bacteria. The bacteria was cultured in two solid media. Bacteria on the Acetobacter media had a resistance in a solution containing alcohol with a higher concentration. Acetobacter was cultured on solid and liquid media for measuring at various ethanol concentrations (0.01%-3%) with different absorbance value. Bacteria with absorbance value 0.500 had the highest oxidation current peak. Later, it was used as bioreceptor for measuring ethanol by cyclic voltammetry method. The linearity of measurement later was compared with spectrophotometric measurement. By using cyclic voltammetry the linearity had higher R2 value than spectrophotometric method. However, it only had stability for two days, there fore it needs more improvement for lasting its lifetime. Basically, electrochemical method has potency for further being improved fur as an alternative method of ethanol on-site measurement.


Introduction
Alcohol, cormercially sold as ethyl alcohol, has been applied to many kinds of goods because of its beneficial properties, such as volatile, good solvent, and less toxic.It also has been applied to many kind of foods and bevearages production [1].However, its usage on foods and bevearages industry should be put into consideration when dealing with islamic halal codification.As fatwa of Ulema Council of Indonesia (Majelis Ulama Indonesia; MUI) No. 4/2003, all the fermented foods or bevearages which have more than 1 % of ethanol are considered as haram [2].So, for protecting the consumers, especially muslims, from being exposed of haram products there should be a method to detect alcohol in foods or beveareges easily on site.
Several methods have been developed, like colorimetry [3].However, this method could be interferred by a simmilar analyte which has the same wavelength absorption of light.Hence, Electrogenerated chemiluminescence (ECL), a sophisticated method for measuring ethanol by applying a simple optical adjustment, was made.Therefore, it provides a high flexibility and able to detect the analyte at the trace concentration with wide range of linearity and low background noise effect [4].However, this method should use the pure enzyme for conducting the measurement.So, it deals with cost and also time consuming.
Biosensors have been become a research interest due to its high sensitivity and selectivity which is very important for applying it in fermented foods and bevearage industry quality control.
Consequently, the bioreceptor, a crucial component of biosensor for detecting the analyte, should be explored to produce a more sensitive and selective biosensor.In this case, an enzymatic process can be a tool for it.Two kinds of enzyme which are involved into an enzymatic reaction with ethanol have been proposed: alcohol dehydrogenase (ADH) and alcohol oxidase (AOX).These enzymes could be easily produced by the microbes.So, rather than extracting the enzymes out from the microbes, it would be cheaper for immobilizing the microbes directly onto the electrode.Thus, it produces a microbe biosensor which has at least two advantages: durable and cheapIn addition, microbe has an ability to reproduce itself in higher rate via cell culture.It is also able to be manipulated easily and has better stability [5].
Previously, we had a research about alcoholic biosensor by using Bacillus sp. as bioreceptor and measured by cyclic voltammetry.However, the result was not optimum because the bactery's amount was unknown and different for each measurement (unpublished).So, it produced a big measurement inconsistency when measuring alcohol above 5%.Based on this result, we decided to produce AOX enzyme from Acetobacter sp. in apple juice and conducting research by voltammetry cyclic.Carbon paste electrode was used as the working electrode and also for immobilizing bacteria.
We also compared the ethanol measurement using UV-VIS and this developed biosensor.

Media Preparation for Growing Bacteria
There were two type of media, the solid and liquid form.The solid form of media was made in two types: Acetobacter and Heterotrof.Acetobacter media was made by mixing 0.5 g yeast extract, 0.3 g peptone, 2.5 g mannitol, and 1.5 g Bacto Agar and put into 300 mL Erlenmeyer 300 flask, then dilluted with 100 mL aquadest.Heterotroph media was made by mixing 1.5 g Bacto Agar, 1.5 peptone, 0.3 g triptone, 0.5 g NaCl, and 0.25 g K2HPO4 into 300 mL Erlenmeyer flask and dilluted with 100 mL aquadest.Those media were put into microwave until dilluted homogenously.After the solutions were ready, the mouth of Erlenmeyer flask were closed by cotton covered by aluminium, later covered again by plastic wrap.Those media than were put into autoclave for 90 minutes then poured into several warmed sterilized petri dishes.Liquid media for growing bacteria was made from Acetobacter media.with the same ingredients and procedure as making solid Acetobacter media before but without addition of Bacto Agar.

Bacteria Preparation
Bacteria were isolated from 2 mL apple juice, which is containing 1%, 2%, 5%, and 8% ethanol,into different eppendorf tubes, and let it fermented for one night.Later the solution was taken and scratched onto solid media with sterilized loopful, then incubated at 37 o C for one night.
The colony which was grown on the media later was moved into phosphate buffer containing 8% for one night.Later, the solution was scratched again onto the other solid agar media.After having the isolated bacteria, later it is moved onto another petri dish containing solid agar media by scratching it.The dishes were wrapped for preventing contamination and stored at 37 o C for one night.The colony on the solid agar media then was moved into liquid agar media and incubated for one night at 37 °C and shaken at 90 rpm inside shaker water bath.The incubated bacteria later was moved again into the new liquid agar media with the same procedure as stated before for 3-4 hours only.All the pocedures above were performed inside a steril laminar column.
The bacteria later was measured for its OD600 absorbance.The harvested bacteria were dilluted into 3 types of concentrations which have absorbance values: 0.250, 0.500, and 0.750.Later, those solutions were stored into different eppendorf tubes and separated by centrifugation for 10 minutes (10,000 rpm, 4 °C).The formed pellets later was separated and washed with aquadest using vortex and centrifugated again.The washing process was repeated twice, then suspended into 0.7 mL phosphate buffer (0.05 M pH 6.8).For counting its density, we used spectrophotometer UV-Vis at the wavelength of 600 nm.The given absorbance value was 0.1 equal to 1-2 x 10 6 cell mL -1, and it should be measured before its usage.

Electrodes Preparation
Into the mortar ferrocene and graphite were mixed homogenously and added with 1 mL of diethyl ether.Those mixture later was mixed again with paraffine until it formed carbon paste.The paste then was put into the small tube compartment, pressed, and cleaned with tissue.The electrodes then were incubated for 7-12 days.After that, all the electrodes were characterized by using K3Fe(CN)6 inside KCl solution.The carbon paste electrodes were made into 6 different compositions of graphite (mg) : paraffin (mg) : ferrocene (mg):

Ethanol Measurement
For electrochemical measurements we used a computer installed with eDAQ potentiostate and Echem v2.1.0as the software.The applied electrodes were: (a) carbon paste electrode as working electrode; (b) Ag/AgCl as refference electrode; and (c) platinum as auxiliary electrode.The measurement parameters were set as follows: Mode : Cyclic EInitial : 0 mV EFinal : 0 mV Rate : 200 mV/s Step W : 20 ms EUpper : 1000 mV ELower : -200 mV Blank current response was observed by putting 5 mL phosphate buffer solution (0.05 M pH 6.8) in the vial.The selected bacteria were proliferated for further analyses with the same former steps.Different ethanol concentrations were used in this analyses: 0.01%, 0.1%, 0.5%, 1%, 1.5%, 2%, and 3%.The electrochemical measurement on the selected bacteria and in the whole ethanol concentration range.
Spectophotometry analyses was begun by searching the maximum absorbance wavelength of ethanol.In this analyses we used 2 cuvettes, the first one was for phosphate buffer solution, and the other one is for the samples.The measurement used the same concentration as the electrochemical measurement method.Stability measurement was conducted in by comparing two kind of electrodes, a disposal type and undisposal one.Each type of electrode is measured for current produced.

Bacteria and electrode preparation
Isolated bacteria were obtained from apple juice.This action was performed for harvesting AOX enzyme from Acetobacter which is living in the extract.Thanks to the ambient condition in the apple juice, it provides Acetobacter life's needs [6].The isolate was reserved with 8% alcohol for make i adaptable to the higher ethanol concentration.The obtained bacteria via scratching method showed that the bacteria survived (Figure 1a).By the result, there was a trend that the more ethanol presence in the solution, it inhibited bacterial growth.From the bacterial screening profile, it was shown that the colony was separated well in Ap4.These bacteria colonies, which were marked on red circle, later would be planted on different solid media, Acetobacter and Heterotroph media.Different composition of electrodes had different peak current (Figure 1b).By using KCl and K3[Fe(CN)6] we screened the elctrode to determine the best composition of modified electrode.The best electrode has a characteristic having a high oxidation peak, because it can provide a wide range of linearity.Based on the result, the electrode composition of 70:30:3.5 had the highest produced current (0.26 x mA for oxidation and -0.44 mA for reduction current respectively).Thus, we chosen this composition for further analyses.Those two isolates later being measured using voltammetry method to determine which kind of media would produce the higher current.For this aim, we need to immobilize the bacteria onto the modified electrode.The immobilization process was simply by absorbing the bacteria onto the electrode surface.On Figure 2 it is shown that Acetobacter media had a higher oxidation current compared to Heterotroph media in both cases.The bacteria in Acetobacter media gave an increasement of oxidation current parallel to the increasement of the ethanol concentration.In other hand, the bacteria from Heterotroph media could not give higher current after ethanol concentration increasement.This indicates Acetobacter media is a better media for harvesting bacteria from apple juice.Hence, we chosen to use Acetobacter media for growing the bacteria from apple juice.Different absorbance were set to 0.250, 0.500, and 0.750 creating 3 levels of cells concentration.These levels were made for differing the amount of bioreceptor on biosensor.Each absorbance value corresponded to 2.5-7.5 million of cells.However, not all cells were immobilized, for each cells concentration the immbolized cells were around 19, 36, and 56 thousand bacteria cells.It means that the more concentrated solution would give more thick immobilized cells on electrode.With increasing ethanol concentration, it was shown that the current getting higher as well (Figure 3a) as the the solution which had absorbance value 0.500 was the highest for both extreme points of linearity test.At the lowest ethanol concentration (0.01%), it gave current as much as 1.8 μA,while the highest ethanol concentration (3.00%) produced 5.609 μA (Figure 3b).

Bacteria concentration and oxidation current
Biosensor is preferred for having high linearity between change of analyte concentration and the response signal.By comparing the R 2 value it was obtained that 0.500 absorbance value gave  higher R 2 value (Figure 4).It means, by using this concentration at absorbance value 0.500, it would produce more linear model.Thus, we can easily determine alcohol concentration without any big influence of residual error.Thus, this result would be compared with the measurement result from spectrophotometry method (Figure5).By comparing R 2 value it could be concluded that the proposed biosensor method had lowerer error compared to the spectrophotometer method, 96.38% compared to 94.07% respectively.However, the error difference was slight, so it needs more technique to enhancing biosensor for obtaining better measurement result.However, it only has low stability.Asharp decrease current produced happened at the second days after the fabrication (Figure 6, day-1 to day-3).It implies the stability of immobilization needs to be improved, or also the way for preserving it.

Discussion
There are several kinds of enzyme which are able to be used for detecting the presence of alcohol.Two kinds of the enzymes are alcohol dehydrogenase (ADH) and alcohol oxidase (AOX).Both of this enzymes utilize alcohol as the energy source for transforming it into other compounds.Both enzymes are grouped into oxidoreductase enzyme.However, ADH takes the reversible reaction comparing to AOX.In addition, ADH needs coenzyme nicotinamide adenine dinucleotide (NAD + ) for catalyzing primer alcohol to aldehyde reversibly as this reaction follows 7 : (a) 70:30:0.7;(b)70:30:3.5;(c) 30:10:0.3;(d) 30:10:1.5;(e) 100:100:1; and (f) 100:100:5.

Figure 6 .
Figure 6.Stability profile of each type of biosensor measurement.