Development of Biological Oxygen Demand Biosensor for Monitoring the Fermentation Industry Effluent

A biosensor was developed for the determination of BOD value of fermentation industry effluent. The developed biosensor was fabricated by immobilizing the microbial consortium on cellulose acetate (CA) membrane in close proximity to a DO probe electrode. The microbial consortium was harvested from the fermentation industry effluent. The BOD biosensor was calibrated by using a solution containing the equivalent amount of glucose/glutamic acid (GGA) as a standard sample solution. The response time was optimized by immobilizing different concentrations of cell biomass on CA membrane. Once the response time was optimized, it was used for determination of BOD of fermentation industry effluent. For analysis of fermentation industry effluent, the response time was observed 7 minutes with detection limit 1 mg/L. Good linear range with GGA standard solution was observed, R 2 0.99 with relative standard deviation (RSD) <%. The observed BOD value by biosensor showed a good comparison with the conventional method for the determination of BOD.


Introduction
Biochemical oxygen demand (BOD) is one of the most important and widely used parameters for characterizing the organic pollution of water and wastewater, which is estimated by determining the amount of oxygen required by aerobic microorganisms for degrading organic matters in wastewater. Conventional BOD method is the well-known BOD 5 which needs 5-day incubation at 20 ∘ C in the dark [1].
e United States includes BOD effluent limitations in its secondary treatment regulations. Secondary sewage treatment is generally expected to remove 85 percent of the BOD measured in sewage and produce effluent BOD concentrations with a 30-day average of less than 30 mg/L and a 7-day average of less than 45 mg/L. e regulations also describe "treatment equivalent to secondary treatment" as removing 65 percent of the BOD and producing effluent BOD concentrations with a 30-day average less than 45 mg/L and a 7-day average less than 65 mg/L [1]. Most pristine rivers will have a �ve day carbonaceous BOD below 1 mg/L. Moderately polluted rivers may have a BOD value in the range of 2 to 8 mg/L. Municipal sewage that is efficiently treated by a three-stage process would have a value of about 20 mg/L or less. Untreated sewage varies but averages around 600 mg/L in Europe and as low as 200 mg/L in the US, or where there is severe groundwater or surface water in�ltration. e generally lower values in the US derive from the much greater water use per capita than in other parts of the world [2].
1.1. Calculation of BOD Value. e BOD of a waste water can be de�ned as the amount of oxygen expressed in milligrams per liter required by the microorganism for the biodegradations of the degradable carbonaceous organic matter present in the water through their biochemical, bioprocess, and under the following reaction conditions: temperature 20 ∘ C, �veday retention time, and darkness to avoid the presence of microscopic algae that produce oxygen by photosynthesis thus interfering with the result. Because the saturation conc. for oxygen in water at 20 ∘ C is approximately 9 mg/L dilution of the sample with BOD free, oxygen-saturated water is necessary to measure BOD values greater than just a few mg/L. BOD of a diluted sample is calculated as where DO I and DO F are initial and �nal dissolved oxygen concentrations (mg/L) and is the decimal fraction of the sample in 300 mL bottle [3]. Biochemical oxygen demand (BOD) is an important index for monitoring organic pollutants in water. e conventional standard method (�ve day BOD test, BOD 5 ), however, is a complicated and a time-consuming procedure, including a �ve-day incubation, and also requires considerable experience and skill to get reproducible results [4]. Fast determination of BOD could be achieved by biosensorbased methods. A common feature of these sensors is that they consist of a microbial �lm that can biooxidize the organic substrate to be quanti�ed, sandwiched between a porous cellulose membrane and a gas-permeable membrane as the biological recognition element. First BOD biosensor was developed by immobilization of Trichosporon cutaneum on the oxygen electrode [5]. Some BOD sensors have been developed and marketed by various manufacturers in both, bio�lm and bioreactor-type con�gurations. Most commercially available BOD sensors are �ow-type systems that can be more easily automated but generally require high maintenance to prevent fouling and clogging [6]. e response is usually a change in concentration of dissolved oxygen or other phenomena such as light emission [7].
Despite the good agreement between biosensor results and conventional BOD analysis, and despite the short response time of biosensors, current BOD biosensor systems still present a series of limitations that restrict their industrial applications: the lack of standardization and legislation in most countries, complicated maintenance requirements, and insufficient resistance to various toxic compounds such as heavy-metal ions, CN − ions, and phenol in the wastewater. It is possible to eliminate the toxic effects of heavy-metal ions by using a chelating agent that complex the ions, for example, ethylene diamine tetra-acetate (EDTA) and sodium diethyl dithiocarbamate [8,9]. Prevention of contamination by other microbes is also important for a reliable bio�lm-type BOD sensor [7].
Literature survey reveals many biosensors developed for BOD determination by using the different type of biological components and also different strategies of immobilization techniques. Qian and Tan [10] used heat-killed B. subtilis for BOD determination and stability of their operation is about 140 days. e immobilized Pseudomonas putida bacterium membrane was placed on the top of an optode, which was linked to a photo diode that detected �uorescence signal. e response time was 15 min for chloride up to 1000 mg/L [11]. Many BOD biosensors have been developed for the determination of high BOD values in industrial wastewater and not adapted to the measurement of low BOD values. An optical �ber biosensor was developed for the evaluation of low BOD values in river waters [12]. BOD sensor system of �ow injection mode, constructed by combining an immobilized microbial reactor with an electrochemical �ow cell of three electrodes con�guration, has been developed to estimate BOD [13]. BOD sensor based on immobilizing multispecies BOD seed for wastewater monitoring has been developed in the �ow system [14]. A novel reactor-type biosensor for rapid measurement of BOD was developed, based on using immobilized microbial cell (IMC) beads as a recognition bioelement in a completely mixed reactor [15]. Some online system for determining organic pollutants by using bio�lm-reactor-based approach [16]. A BOD biosensor based on the microbial fuel cell principle was tested for online and in situ monitoring of biodegradable organic content of domestic wastewater [17]. Some other kind of biosensor was developed for the amperometric short time BOD analysis by applying micro�uidic respirometer [18]. A biosensor was developed for the determination of BOD value of speci�c industry effluent such as for meat industry effluents [19], paper and pulp industry effluents [20]. Instead of bacterial cell some yeast cells were also used for the construction of BOD biosensor [21].
In this study, we propose a new analytical approach that has very cost-effective and simple idea for determination of BOD value of fermentation industries effluents. is new approach utilizes microbial consortium that was isolated from fermentation industry effluent and separately immobilized on the cellulose acetate membrane. e primary objective was isolation of microbial consortium, immobilization on transducer, that is, DO probe, optimization of response time, and comparing BOD by using this biosensor with Winkler's method (BOD 5 ).

Chemical Reagents.
Phosphate buffer was prepared by dissolving (KH 2 PO 4 8.5 g, K 2 HPO 4 21.75 g, Na 2 HPO 4 33.4 g, NH 4 Cl 1.7 g) in 1 L water, ferric chloride (FeCl 3 ⋅6H 2 0) 0.25 g/L, calcium chloride (CaCl 2 ) 27.5 g/L, magnesium sulphate (MgSO 4 ⋅7H 2 O) 22.5 g/L dilution water was prepared by adding 2 mL of each of the above reagent. Some other chemical reagents as manganese sulphate solution (MnSO 4 ⋅4H 2 O), alkaline-iodine reagent (dissolved 500 gm of NaOH and 135 gm of NaI or 700 gm KOH and 150 gm KI), were added in distilled water and diluted to 1 liter. Add 10 gm of NaN 3 dissolved in 40 mL of distilled water), concentrated H 2 SO 4 , starch (1 gm of starch was dissolved in 100 mL distilled water), standard sodium thiosulphate (0.025 N dissolved exact weight of 9.205 gm anhydrous sodium thiosulphate in distilled water and diluted it to 1 liter), and Nutrient Broth M002 (HiMedia, Mumbai). All chemicals were purchased from Sigma (St. Louis, USA) and used in analytical or higher grade. Cellulose acetate membrane (0.2 OE-66) was purchased from Whatman part of GE Healthcare, India.

2.2.
Apparatus. DO sensor amperometric gold/silver membrane type (DO range 0 to 40.0 ppm and temperature range 0 to 500 ∘ C) and its Resolution accuracy (DO 0.1 ppm, Temperature 0.1 ∘ C), and temperature sensor RTD (PT-100) make of Labtronics Inc., Ontario, Canada.

Procurement of Industrial Effluent and Its Characteristics.
Fermentative industry effluent was procured from Patiala distiller and manufacturers situated at Village-Main, District-Patiala, Punjab (India), and characteristic features of industrial effluents are given in Table 1.

Isolation of Microbial
Consortium. e microbial consortium isolated in nutrient broth medium (readymade) by using 2% inoculums (effluent from fermentative industry) was used. Aer inoculation the culture medium was kept on shaker incubator at 37 ∘ C for 24 hours. Subculturing was done again in nutrient broth at 37 ∘ C for 24 hours, and culture was maintained in the same medium every week and 2% of inoculum was used.

BOD Determination by Conventional (Five-Day) Method.
e �ve-day BOD of sample was determined by �inkler's method [22]. Calculations: DO mg/L = Hypo 8 1000 Volume of sample (2) = volume of Na 2 S 2 O 3 used, = normality of Na 2 S 2 O 3 (Hypo) (0.025N), 8 = equivalent weight of oxygen. e test sample was prepared by diluting the I.F. and in water has free of oxygen and microbes, and the �nal test sample 10% of I.F. All experiments were done in triplicate, and their average values are considered.

Operation and Calibration of DO Probe.
Oxygen dissolves in water, oen referred to as DO; the main source of DO in water is diffusion from air and photosynthetic activity. Nonpolluted surface water is normally saturated with DO. First, temperature was set according to solution temperature by "temperature knob. " Place the DO probe in 2% sodium sulphite solution (solution having no oxygen because it's absorbed by sodium sulphite), allow the display to attain equilibrium, and then set the zero by "zero knob. " It calibrated the instrument with known value of DO solution (fully agitated with oxygen-distilled water at different temperature) that was used ( Table 2).
Hold DO probe in �ask containing saturated distilled water and agitate the water and if necessary adjust the meter reading with "cal" knob. Now instrument was ready to determine the DO of unknown solution.

Immobilization of Microbial
Consortium on CA Membrane. e basis of a microbial biosensor is the close contact between microorganisms and the transducer. us, fabrication of a microbial biosensor requires immobilization on the transducer with a close proximity. Since microbial biosensor response, operation stability, and long-term use are, to some extent, a function of the immobilization strategy, the immobilization technique is the physical method in which membrane entrapment of whole cell was achieved by using cellulose acetate membrane. First, the cells were harvested from a culture medium by centrifuge at 6000 RPM, then suspension was made in the phosphate buffer pH 7.4 that showed OD 1.0 at 600 nm. At this stage the cell concentration was found to be 1 10 8 cfu/mL. Further this cell biomass was used for immobilization on cellulose acetate membrane at different cell concentration.

Calibration of BOD Biosensor and Response Time
Optimization. BOD biosensor was calibrated using standard solution having the equivalent amount of glucose and glutamic acid (GGA) 150 mg/L, with BOD value of 220 mg/L ± 11.0. Optimization of response time, that is, 0, 4, 8, 12, 16, 20, 24, 28, and 30 minutes for microbial degradation of the equivalent amount of GGA solution by the microbial consortium was studied. Aer response time optimization, the linear range of BOD biosensor was also studied by using different concentration range (1-150 mg/L) of GGA standard solution. 5 . 100 mL diluted sample was taken, an initial DO was found by dipping the oxygen electrode and aer that oxygen, electrode was coupled with cellulose acetate membrane containing immobilized microbial consortia and found the �nal DO aer a particular time (response time), and calculated the BOD value of respected sample (fermentation industry effluent). e BOD value observed by biosensor was also compared against the BOD value determined by the conventional method of same sample. Fermentative I.E. BOD (mg/L) BOD 5 BOD biosensor 5% 180 ± 12 170 ± 8 10% 180 ± 10 190 ± 6 .

Estimation of BOD Value of Effluent
Sample. e �ve-day BOD of sample was determined by Winkler's method. e value of BOD of fermentative I.F. determined by conventional method was found 180 ± 10 mg/L (Table 3).

Optimization of Response
Time. e response time (time taken by microbial cell to oxidize the GGA solution) was studied by using different volumes of cells (microbial consortia) biomass at absorbance = 1, that were immobilized on cellulose acetate membrane. Each immobilized membrane was coupled with the probe and tested against the GGA standard solution, and decrease in DO was observed (Table 4). e response time with cell biomass of microbial consortium concentration 2 × 10 7 cfu/mL was observed only 7 minutes (Figure 1).
König et al. [23] developed BOD biosensor for determination of BOD for nitri�cation (N-BOD) by using nitrifying bacteria immobilized at an oxygen electrode, and response time was about 12 minutes. Microbial fuel cell (MFC) was used in the determination of BOD value of waste water; the minimum detection limit of this MFC is about 0.2 mg/L with a response time of two hours [24]. Chen et al. [25] developed BOD biosensor that was also developed with response time of 15 minutes and also found linear relationship between the response (sensor current) and BOD values ranging from 10-15 mg/L. At higher cell concentrations, the response time was again increased, because the layer of immobilized cell on membrane is thick, and they cause a low rate of consumption of organic substances. Cheng et al. [26] used luminescent bacteria Vibrio �scheri� �hotobacterium phosphoreum� and recombinant Escherichia coli as potential indicators of BOD in the domestic waste water and response time for biosensor 90, 120, and 150 minutes, respectively. In the present study, the linear range of developed BOD biosensor was also observed with different concentration of standard GGA solution. We found a good correlation coefficient ( 2 ) about 0.99 with RSD < 9%, and also the detection limits were found 1 mg/L ( Figure 2).

Comparison of BOD and BOD Estimated by Biosensor.
Biosensors constructed by using microbial consortium were tested for determination of BOD value of fermentative I.E. As shown in Table 5 relatively good agreement between the two methods was obtained for the test sample with relative error ± (6-8). BOD biosensor has also shown good agreement between the results of the sensor BOD measurement and those obtained from conventional BOD analysis [27].
e experiments were done in triplicate, and the average values are given. e developed BOD biosensor is reliable, economical because here we use the microbial consortium that was isolated from the fermentation I.F., and one advantage of this biosensor is their wide range of application for different types of waste water because the microbial consortia are capable to degrade an extensive range of organic pollutants present in waste water. Earlier developed BOD biosensor based on speci�c microorganism has major disadvantage regarding their narrow range of degradation of organic compound [14].

Conclusion
In this study, a microbial electrode biosensor consisting of immobilized living whole cells on cellulose acetate membrane and oxygen probe has been developed for the estimation of biochemical oxygen demand (BOD). Immobilized microbial consortium isolated from the fermentation industry effluents was employed for the microbial electrode sensor for BOD. is BOD biosensor was calibrated by using a solution containing the equivalent amount of GGA (BOD = 220 mg/L) as a standard sample solution and optimized the response time. Once response time optimized, it was used for determination of BOD of fermentation industry effluent sample. e response time of microbial consortium was very fast only 7 minutes to give BOD value of fermentation industrial effluent. BOD value of fermentation industry effluent was also determined by conventional 5-day methods. e comparison of both BOD value, one BOD value from BOD biosensor and other from the conventional 5-day methods, shows good comparable results.