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Quick and reliable estimation of BOD load of beverage industrial wastewater by developing BOD biosensor
Sensors and Actuators B 133 (2008) 478–483
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Quick and reliable estimation of BOD load of beverage industrial wastewater by developing BOD biosensor Purnima Dhall a , Anil Kumar a , Abha Joshi a , Tushya Kumar Saxsena c , Angamuthu Manoharan b , Santosh Dayal Makhijani b , Rita Kumar a,∗ a b c
Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India Central Pollution Control Board, Parivesh Bhavan, East Arjun Nagar, Delhi 110032, India National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012, India
a r t i c l e
i n f o
Article history: Received 20 October 2007 Accepted 5 March 2008 Available online 21 March 2008 Keywords: BOD biosensor Immobilized microbial membrane Biochemical oxygen demand Beverage wastewater
a b s t r a c t An amperometric biosensor for determination of biochemical oxygen demand in wastewater has been developed to overcome the time consuming monitoring procedures. The performance and stability of the immobilized membrane have been investigated at 37 ◦ C and pH 6.8. Immobilized microbial membranes maintain their stability and activity after intermittent use for 400 cycles when stored at 4 ◦ C in sodium phosphate buffer pH 6.8. The response time of the BOD sensor was only 90 min, being independent of the concentration, and the lower detection limit was 1 mg/l. The obtained BOD values showed correlation with that of the conventional method for BOD determination (BOD5) with a deviation of ±10%. Moreover, the sensor exhibits good repeatability (3.39–4.45%) and reproducibility (1.85–2.25%). Software was added to upgrade this sensor and to make it a promising candidate for online monitoring. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Water pollution requires more attention, and monitoring of water quality is an important aspect of wastewater management. Water quality is evaluated in terms of organic matter present in it which is usually calculated before the discharge of wastewater in to river water stream. Regular monitoring of BOD discharge is required at all publicly owned industrial wastewater treatment plants. Biochemical oxygen demand is most widely used parameter for such estimation and requires minimum of 3–5 days and it’s a universal method for measuring organic load. Results of BOD tests cannot be obtained immediately, so they can be hardly used as feedback data for process control of wastewater treatment or as the data proving legal observance of effluent water quality. Many studies aiming at shortening the measuring period of BOD were conducted for a long time [6] until microbial BOD sensors were developed to meet such shortcomings. Determination of BOD load in short time is now possible with the help of microbial BOD sensors. Since the advent of a microbial BOD sensor in 1977, when Karube discovered a rapid alternative for sensing BOD load [7], many research
∗ Corresponding author at: Institute of Genomics and Integrative Biology, 506, Environmental Biotechnology Division, Mall Road, Delhi University Campus, Delhi 110007, India. Tel.: +91 11 27666156/147; fax: +91 11 27667471. E-mail address: [email protected] (R. Kumar). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.03.010
reports have been published with modifications occurring at every step to circumvent the lacunae faced by the previous authors. Lots of efforts have been put henceforth for the development of BOD biosensors which of late include the determination of BOD load in real wastewaters [11,19,14–17,10,4,5] but still there remains a number of lacunae viz, the capability of sensor to meet the varying BOD load of wastewater being emanated from any industry and practical feasibility of the BOD biosensor to determine the BOD load of wastewater as and when it comes to the laboratory (off-line) or online. This is possibly due to the fact that BOD determination is multifactorial and until and unless all the parameters are taken into account, it will not be possible to reach the desired goal. Biosensors have passed the point of mere combinations of biological components with transducing elements and now practical applications must be realized. Though few BOD biosensors are in the market for the purposes [9] but as of now there does not seem to be any ideal biosensor which has real reproducibility for varying loads of pollution in beverage wastewater and which is capable of determining the BOD load instantly. In the present scenario based on existing lacunae, an attempt was made to develop an ideal device (BOD biosensor) having a computer-aided software capable of determining the varying BOD load of beverage wastewaters which facilitated the instant monitoring. This would help in the real sense, in determining the efficiency of treatment systems and instant actions on the same thereof. The developed BOD biosensor was extensively studied for beverage wastewater collected over a period of different
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times. The developed biosensor has advantages of determining the BOD load of beverage wastewater within 1 h and giving BOD values of the order comparable to conventional BOD test irrespective of the varying load of wastewater due to different operations occurring in the industries. The sensor requires using 60 mg/l of GGA every time the sample needs to be analyzed. The sensor response time is 15 min; baseline recovery takes 15 min and after analyzing only two/three samples, the BOD results can be obtained with reliability. The membrane is stable up to 400 days with pre-conditioning at various intervals. 2. Experimental 2.1. Chemicals and multimeter Charged nylon membrane (SIGMA) with a pore size of 0.45 m was used throughout the investigation. d-Glucose and d-glutamic acid were obtained from Sigma, Germany. Whereas, the other chemicals used to prepare the growth medium were procured from Hi-Media, India, Multimeter (Model 2700) was purchased from Keithley Instruments Inc. 2.2. Bioreceptor and immobilization of microbial membrane A synergistic microbial consortium, pre-tested as a reference seeding material in BOD analysis [2,12] was used as a bioreceptor. The adsorption method was used for the immobilization of microbial consortium on nylon membrane [14]. The microbial consortium were harvested in their log phase of growth, cells were washed three times with sodium phosphate buffer pH 6.8 by centrifugation. Nylon membrane was placed on membrane filter and 500 l of the cells were filtered through the membrane and then membrane was left to dry for 18–20 h at room temperature. 2.3. Pre-conditioning of the membrane The immobilized membrane was prepared by filtering the calculated amount of microbial consortia through nylon membrane. The microbial membranes were activated by immersing the membranes in sodium phosphate buffer (disodium hydrogen phosphate and sodium dihydrogen phosphate) containing different concentrations of glucose–glutamic acid (GGA 1920–12,000 mg/l) and left to stand at 4 ◦ C till further use (Table 1). The pre-conditioning was done by stirring. It was observed that the preconditioning of membrane with 60 mg/l of GGA for 2 h while stirring activates the membrane fully to be used for sensor measurements. A number of membranes were prepared at different times but with same known consortia (mentioned above) immobilized on nylon membrane and studied for their reproducibility as well as for their shelf life. 2.4. Assembly of the microbial sensor and measurement of response The sensor response was measured by coupling the immobilized membrane to the cathode of the oxygen probe. A Clark type probe for dissolved oxygen was used as the physical transducer, which Table 1 Different concentrations of GGA S. no. 1 2 3 4
Concentration of GGA 1,920 3,840 7,680 12,000
479
consisted of a platinum cathode as the working electrode and a silver anode as the reference electrode. The working and reference electrodes of the BOD sensor were connected to a potentiostat and the output current was recorded through a multimeter, which amplified the response to nA. An applied potential of −0.650 mV versus Ag/AgCl was delivered to the Pt-working electrode throughout all the measurements (Fig. 1). The BOD sensor measurements were made using steady state method. The steady state indicates the consumption of oxygen by microorganisms which diffuses from a sample solution to the membrane at equilibrium. In steady state method, the difference of current between the two steady states reflects the respiration rate of substrate and was used for BOD sensor estimation. The measuring time was 10–15 min followed by 15–16 min recovery time. In order to stabilize the BOD sensor system and measure the response of the immobilized microbial membrane, 100 ml of phosphate buffer (50 mM) pH 6.8 was placed in a thermo stated cell at 35 ± 2 ◦ C under constant and moderate stirring. Aliquots of stock synthetic/industrial samples were added after a stable current was attained for 30 min (initial steady state current). The current change (decrease) was observed after addition of the samples until a steady state was reached. The response was calculated on the basis of current difference between the initial steady state current and final steady state current. 2.5. Calibration Since GGA is used as a reference standard to standardize conventional 5-day BOD measurement, all the BOD measurements with the sensor in this study were carried out using these GGA solutions. A stock solution containing equal volume of 150 mg/l of glucose and glutamic acid with an assigned 5-day BOD value of 220 mg/l was prepared [1]. Different concentrations of GGA ranging from 5 to 120 mg/l were used for the calibration of the BOD by conventional method as well as by biosensor. 2.6. Wastewater samples Real wastewater samples, comprising both inlet as well as outlet samples, were procured over a period of time at different intervals, from a soft drink company of International repute, located near New Delhi. The pH value of the wastewater should be in the range of 6.6–8.5, whereas BOD should be around 30 mg/l before it is discharged outside. Wastewater characteristics of beverage wastewater included BOD in the range of 316 mg/l–2890 mg/l, and COD in the range of 605 mg/l–6778 mg/l. 2.7. Conventional COD and BOD 5-day test The COD and 5-day BOD tests of standard solution and all effluent samples were carried out according to the method described in Standard Methods for Examination of Water and Waste-waters [1]. 2.8. Measurement of BOD by biosensor The biosensor was used to estimate the BOD of a large number of beverage wastewater samples. The dilutions had to be calculated manually for each sample so that the BOD value falls within the range followed by the calculation of BOD. 2.9. Need for the development of software It was likely that by carrying out some more improvements and modifications, it may be possible to reach to a conclusion for the commercialization of the sensor for beverage industry. Some of the
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P. Dhall et al. / Sensors and Actuators B 133 (2008) 478–483
Fig. 1. Schematic diagram of the amperometric biosensor.
aspects that were considered for achieving the above were as follows: • Automated generation of the dilution required for the estimation of BOD. • DO correction factor for the instrument. 2.10. Software development Software, “BioSensBOD ” was developed on visual basics platform meant for graphical displays, BOD calculation, online data acquisition and other calculations. The developed software “BioSensBOD ”, which was developed on visual basic platform, has got two main parts: (A) In first part, users enter the parameters like sample description, membrane ID, % dilution, maximum time for taking readings, time interval between readings, and output file name in the form “parameters” shown in supplementary Fig. 1. Thereafter it jumps to the next form “Online Graph Plotting” (supplementary Fig. 2). This form has got four command buttons i.e. Start, Stop, Print and Exit. By pressing Start button, program starts acquiring data from the Keithley Multimeter (Model 2700) through RS232C interface and the date and time of starting of the acquisition of the data is displayed on the screen. The acquired data is also saved in the user defined output file. After the maximum time, the plotted graph can optionally be printed on the printer. (B) The second part is offline part. This has got further three parts: (i) membrane data, (ii) BOD calculator and (iii) offline graph plotting. (i) Membrane data: By selecting this user can enter the new membrane reference data at different GGA concentrations along with its ID, modify the existing membrane data, delete any membrane reference data or can also undelete the deleted membrane reference data. The reference membrane data form is shown in supplementary figure (Fig. 3). (ii) BOD calculator: This calculator asks the user to enter the membrane ID, I (i.e. Imax − Imin ) at 60GGA, I (i.e. Imax − Imin ) of the test sample, and the percentage dilution of the sample used. Thereafter, the program compares the present I at 60GGA with the reference I of the selected membrane and calculates the correction factor and applies it for the calculation of BOD level and the same is displayed in mg/l. In case the equivalent I for the sample reaches the saturation level, then program does the reverse calculation and gives the value of the dilution of the sample to
be used for proper measurements. The BOD calculator data is shown in supplementary Fig. 4. (iii) Offline graph plotting: This form asks the user to enter the file name whose data is to be plotted on the screen and then plots the data after reading the file. The user can also view the data of any selected file on the screen. The offline graph plotting form is shown in supplementary Fig. 5a and b. 2.11. BOD estimation of beverage wastewater using developed device The device was assembled by connecting the immobilized electrode to the developed highly stable constant volt source and multimeter which in turn is connected to a mobile PC through RS232C interface. Mobile PC is installed with developed software “BioSensBOD ” built on visual basics platform. This software acquires the real time current data from the multimeter and plots the same in the form of desired graphical display as well as save the same in the user defined file along with the time in seconds. In starting, electrode was dipped in sodium phosphate buffer placed on the magnetic stirrer and external polarization voltage was applied through the highly stable developed voltage source. Current through the electrode was measured by the multimeter. Then stability of the immobilized microbial membrane was observed as displayed by developed software installed in a mobile PC which in turn is connected to a multimeter through RS232C interface. Change in current (i.e. I = Imax − Imin ) was calibrated for different concentrations of GGA. Sample solution was replaced now with fresh sodium phosphate buffer and assembly was stabilized. In order to calculate the BOD, the percentage of the wastewater was added in sodium phosphate buffer, as determined by the software. Finally, BOD value of wastewater was calculated with the help of BOD calculator as mentioned above. This BOD value was compared to BOD5 as determined by titration-based method. 3. Results and discussion The BOD sensor was aimed at being highly capable for analyzing a sample of complex constituents with relatively low selectivity. The developed sensor responded to all kinds of biodegradable organic solutes present in the sample of beverage industrial wastewater. However, the problem remained as to how to determine the BOD load of any wastewater as and when it arrives in the laboratory, i.e. which dilution to use and whether the BOD values determined are
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Fig. 3. Graphical representation showing the comparative performance of biosensor with and without the use of software.
Fig. 2. Response curve of different concentrations of GGA as measured by the sensor: (a) membrane response, (b) linearity of membrane and (c) calibration curve.
comparable with the conventional method or not and how much time is required to determine the reliable BOD values. With this aim in mind, an extensive testing of beverage wastewater was undertaken by conventional method as well as by the developed BOD biosensor. 3.1. Calibration The calibration of the sensor involves correlating the sensor response with the 5-day BOD value of the solution. For calibration, the linearity in response is measured. The linear range of the sensor is defined as the substrate range that gives a signal directly proportional to the concentration. Linearity in response time over a certain concentration range is a measure of the detection capacity to analyze wastewater with varying concentrations. The BOD values of different concentrations of GGA as determined by the developed sensor showed linearity up to 60 mg/l of GGA, which corresponded to BOD values of 45 mg/l. The linearity of this range was satisfactory [R2 = 0.9914, Fig. 2(a–c)]. 3.2. Determination of BOD load of beverage wastewater with the developed software The COD and BOD of beverage wastewater varied from 1000 to 3400 and 300 to 2250 mg/l, respectively, over a period of time (supplementary Fig. 6). Based on the extensive data obtained
(Table 2), a software was designed which instantly gave the actual dilution of the wastewater to be used for the determination of BOD values. The correction factor was taken into consideration while integrating software with the device. Thus, it was possible to determine the BOD values of wastewater instantly after using just one concentration, thereby reducing the time (Fig. 3). The BOD values of inlet and outlet samples of effluent as determined by the developed software aided BOD biosensor are shown in Table 3. The results showed that the BOD values were comparable to BOD values determined by the conventional method and lay well within the range. In this case, COD was not required. Therefore, the determination of BOD load was accomplished in 90 min as against 5 days method without the need of performing COD analysis. The percentage variation in the BOD results was limited to ±8% in case of inlet samples and to ±10% in case of outlet samples. Variation was observed in the ratio of BODsensor :BOD5 in case of real wastewater .This was corroborated to the different measuring principles involved in both the cases as well as due to variability in composition of wastewaters. However, it was predicted by Liu and Mattiasson [9] that it is possible to minimize the problems by selecting suitable microbial strains as biological recognition elements. Rastogi et al. [14] reported that a BOD biosensor based on pre-tested, synergistic formulated microbial consortia was capable to sense the BOD load of a wide variety of industrial wastewaters having low moderate–high biodegradability. It has been emphasized that a pre-selection of microorganisms for their capability to degrade a range of pollutants can serve to develop an ideal BOD biosensor. Such a biosensor can cater to a variety of industrial effluents in determining the BOD load. Earlier, BOD biosensors have been tried for various synthetic substrates [8,10,11,14] but when applied to real wastewaters, they did not give good correlation with BOD5 values. 3.3. Repeatability and reproducibility As per the conventional BOD method, analysis of a GGA solution check with an average BOD5 value of 198 mg/l and a variability of precision of about ±15% is intended to be the reference for any laboratory. In the present study, the repeatability error of 3.49 and 4.45% was obtained in case of GGA (60 mg/l) when two membranes were used (Table 4). It has been reported earlier, the repeatability varies from ±2.4% to ±10% in case of single strain sensors with maximum value of ±10% in case of Pseudomonas putida, ±5% to ±11% for sensors based on the mixture of two identified strains with maximum ±11% in case of mixture of two identified bacteria, Citrobacter sp. and Enterobacter sp., ±1.3% to ±12.4% for those multi-strain based
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Table 2 Percentage deviation of BODs vs. BOD5: beverage wastewater without using software S. no.
COD (mg/l)
BOD5 (mg/l)
Sample percentage used
BOD sensor (mg/l)
Percentage deviation BODsensor vs. BOD5
1
2090
1270
2.75 3.00 3.25 3.50 3.75 7.00 10.00
1382 1256 1167 1086 1013 966 624
+8.82 −1.10 −8.11 −14.49 −20.24 −23.94 −50.87
2
2050
1250
2.75 3.00 3.25 3.50 3.75
1320 1282 1262 1232 1223
+5.6 +2.56 +0.96 −1.44 −2.16
3
1665
1232
3.00 3.25 3.50 3.75 4.00
1502 1456 1328 1245 1229
+21.92 +18.18 +7.79 +1.06 −0.24
4
1456
803
3.50 3.75 4.00 4.25 4.50 4.75
1156 1101 1093 1041 981 946
+43.96 +37.11 +36.11 +29.64 +22.17 +17.81
5
2702
1810
2.00 2.25 2.50 3.00 3.50 5.00 5.50
1960 1797 1752 1584 1486 724 673
+8.29 −0.72 −3.20 −12.49 −17.90 −60.00 −62.82
Table 3 Percentage deviation of BODs vs. BOD5: (a) beverage inlet wastewater and (b) beverage outlet wastewater S. no.
BOD5
BODs
Percentage deviation BODs vs. BOD5
BODs/BOD5
(a) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
610 460 790 810 630 520 660 729 826 1098 410 2250 300 777 1820 1245
605 449 783 878 626 543 673 710 848 1087 409 2105 295 785 1808 1275
−0.8 −2.4 −0.9 +8.4 −0.6 +4.4 +2.0 −2.6 +2.7 −1.0 −0.2 −6.4 1.7 +1.0 −0.6 +2.4
0.99 0.97 0.99 1.08 0.99 1.04 1.02 0.97 1.02 0.98 0.99 0.93 0.98 1.05 0.99 1.02
(b) 1 2 3 4 5 6 7 8 9 10
58 69 32 48 47 27 54 44 40 45
62 69 33 50 50 28 49 48 42 48
+6.9 −0.0 +3.1 +4.2 +6.4 +3.7 −9.2 +9.0 +5.0 +6.7
1.06 1.00 1.03 1.04 1.06 1.03 0.90 1.09 1.05 1.06
BOD5 estimation by 5-day BOD test BODs estimation by developed sensor.
sensors with maximum in case of heat killed cells and ±1.5% in case of predefined microbial consortia based sensors [8]. In the present study, in a sensor based on predefined, pre-tested microbial consortium, the reproducibility of 1.8% was obtained in case of GGA and of ±1.44% was obtained when real wastewater was applied for detecting the BOD load. Sakai et al. [18] reported the sensors based on magnetic activated sludge having reproducibility within ±5%. However, the determination of BOD of a wide variety of effluents and reproducibility in terms of monitoring of a wide variety of substrates remained a problem. 3.4. Operational stability Number of membranes were tested for operational stability in biosensor and it was found to be more than 400 days (supplementary Fig. 7) but the membrane was required to be conditioned after 120 days (data not shown). It has been reported [14] that a mixture of two identified bacteria gave an operational stability of 60 days with more than 200 measurements. The noncompliance of results with BOD5 was attributed to the presence Table 4 Repeatability and reproducibility of sensor membranes: repeatability of sensor with membrane AI–AVI (n = 12) using GGA S. no.
Membrane
Mean
S.D.
R.S.D.
Standard error
1 2 3 4 5 6
AI AII AIII AIV AV AVI
280.92 364.25 337.50 367.00 354.25 348.92
3.20 3.14 2.87 3.13 3.11 3.37
1.14 0.86 0.85 0.85 0.88 0.97
0.92 0.91 0.83 0.90 0.90 0.97
AI–AVI are same immobilized membranes prepared at different times with same known consortia.
P. Dhall et al. / Sensors and Actuators B 133 (2008) 478–483
of many different microorganisms used in conventional method thus being able to biooxidize a larger range of organic substrates. The operational stability of a sensor is affected to a great extent upon the preconditioning of the membrane. The preconditioning has been defined in two ways one to increase the range of assimilation of substrates by the immobilized microbes [10,14], secondly to fully activate the immobilized microbes in a newly constructed membrane [13]. The activation of the membranes at various periods of time increases the operational stability. Li et al. [3] have reported that fully activated cells remained stable and sustained activity for more than 200 reproducible measurements over a period of two months. The operational stability of our developed sensor is 400 days which is the first report in this regard. 4. Conclusion An automated computer aided software integrated BOD biosensor was developed which is able to determine BOD load within one and half hour. The results of extensive testing of developed BOD biosensor on beverage wastewater over a period of time demonstrate that BOD values obtained are comparable to conventional BOD values, irrespective of varying load of wastewater (which might occur due to different operations occurring in industries). Moreover this shows good reproducibility and repeatability over a period of time. The membrane is stable up to 400 days with pre-conditioning at various intervals. This is the first report on the determination of BOD load of real wastewater, i.e. beverage industrial wastewater in matter of hours irrespective of BOD load at any given time. From above results we can conclude that the developed biosensor is pertinent for online, monitoring. Acknowledgements We acknowledge the Ministry of Environment and Forests, New Delhi, for financial assistance. We are also thankful to Prof. S.K. Brahmachari, Director, Institute of Genomics and Integrative Biology for providing necessary facilities. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.snb.2008.03.010.
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Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Quick and reliable estimation of BOD load of beverage industrial wastewater by developing BOD biosensor Purnima Dhall a , Anil Kumar a , Abha Joshi a , Tushya Kumar Saxsena c , Angamuthu Manoharan b , Santosh Dayal Makhijani b , Rita Kumar a,∗ a b c
Institute of Genomics and Integrative Biology, Mall Road, Delhi 110007, India Central Pollution Control Board, Parivesh Bhavan, East Arjun Nagar, Delhi 110032, India National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110012, India
a r t i c l e
i n f o
Article history: Received 20 October 2007 Accepted 5 March 2008 Available online 21 March 2008 Keywords: BOD biosensor Immobilized microbial membrane Biochemical oxygen demand Beverage wastewater
a b s t r a c t An amperometric biosensor for determination of biochemical oxygen demand in wastewater has been developed to overcome the time consuming monitoring procedures. The performance and stability of the immobilized membrane have been investigated at 37 ◦ C and pH 6.8. Immobilized microbial membranes maintain their stability and activity after intermittent use for 400 cycles when stored at 4 ◦ C in sodium phosphate buffer pH 6.8. The response time of the BOD sensor was only 90 min, being independent of the concentration, and the lower detection limit was 1 mg/l. The obtained BOD values showed correlation with that of the conventional method for BOD determination (BOD5) with a deviation of ±10%. Moreover, the sensor exhibits good repeatability (3.39–4.45%) and reproducibility (1.85–2.25%). Software was added to upgrade this sensor and to make it a promising candidate for online monitoring. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Water pollution requires more attention, and monitoring of water quality is an important aspect of wastewater management. Water quality is evaluated in terms of organic matter present in it which is usually calculated before the discharge of wastewater in to river water stream. Regular monitoring of BOD discharge is required at all publicly owned industrial wastewater treatment plants. Biochemical oxygen demand is most widely used parameter for such estimation and requires minimum of 3–5 days and it’s a universal method for measuring organic load. Results of BOD tests cannot be obtained immediately, so they can be hardly used as feedback data for process control of wastewater treatment or as the data proving legal observance of effluent water quality. Many studies aiming at shortening the measuring period of BOD were conducted for a long time [6] until microbial BOD sensors were developed to meet such shortcomings. Determination of BOD load in short time is now possible with the help of microbial BOD sensors. Since the advent of a microbial BOD sensor in 1977, when Karube discovered a rapid alternative for sensing BOD load [7], many research
∗ Corresponding author at: Institute of Genomics and Integrative Biology, 506, Environmental Biotechnology Division, Mall Road, Delhi University Campus, Delhi 110007, India. Tel.: +91 11 27666156/147; fax: +91 11 27667471. E-mail address: [email protected] (R. Kumar). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.03.010
reports have been published with modifications occurring at every step to circumvent the lacunae faced by the previous authors. Lots of efforts have been put henceforth for the development of BOD biosensors which of late include the determination of BOD load in real wastewaters [11,19,14–17,10,4,5] but still there remains a number of lacunae viz, the capability of sensor to meet the varying BOD load of wastewater being emanated from any industry and practical feasibility of the BOD biosensor to determine the BOD load of wastewater as and when it comes to the laboratory (off-line) or online. This is possibly due to the fact that BOD determination is multifactorial and until and unless all the parameters are taken into account, it will not be possible to reach the desired goal. Biosensors have passed the point of mere combinations of biological components with transducing elements and now practical applications must be realized. Though few BOD biosensors are in the market for the purposes [9] but as of now there does not seem to be any ideal biosensor which has real reproducibility for varying loads of pollution in beverage wastewater and which is capable of determining the BOD load instantly. In the present scenario based on existing lacunae, an attempt was made to develop an ideal device (BOD biosensor) having a computer-aided software capable of determining the varying BOD load of beverage wastewaters which facilitated the instant monitoring. This would help in the real sense, in determining the efficiency of treatment systems and instant actions on the same thereof. The developed BOD biosensor was extensively studied for beverage wastewater collected over a period of different
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times. The developed biosensor has advantages of determining the BOD load of beverage wastewater within 1 h and giving BOD values of the order comparable to conventional BOD test irrespective of the varying load of wastewater due to different operations occurring in the industries. The sensor requires using 60 mg/l of GGA every time the sample needs to be analyzed. The sensor response time is 15 min; baseline recovery takes 15 min and after analyzing only two/three samples, the BOD results can be obtained with reliability. The membrane is stable up to 400 days with pre-conditioning at various intervals. 2. Experimental 2.1. Chemicals and multimeter Charged nylon membrane (SIGMA) with a pore size of 0.45 m was used throughout the investigation. d-Glucose and d-glutamic acid were obtained from Sigma, Germany. Whereas, the other chemicals used to prepare the growth medium were procured from Hi-Media, India, Multimeter (Model 2700) was purchased from Keithley Instruments Inc. 2.2. Bioreceptor and immobilization of microbial membrane A synergistic microbial consortium, pre-tested as a reference seeding material in BOD analysis [2,12] was used as a bioreceptor. The adsorption method was used for the immobilization of microbial consortium on nylon membrane [14]. The microbial consortium were harvested in their log phase of growth, cells were washed three times with sodium phosphate buffer pH 6.8 by centrifugation. Nylon membrane was placed on membrane filter and 500 l of the cells were filtered through the membrane and then membrane was left to dry for 18–20 h at room temperature. 2.3. Pre-conditioning of the membrane The immobilized membrane was prepared by filtering the calculated amount of microbial consortia through nylon membrane. The microbial membranes were activated by immersing the membranes in sodium phosphate buffer (disodium hydrogen phosphate and sodium dihydrogen phosphate) containing different concentrations of glucose–glutamic acid (GGA 1920–12,000 mg/l) and left to stand at 4 ◦ C till further use (Table 1). The pre-conditioning was done by stirring. It was observed that the preconditioning of membrane with 60 mg/l of GGA for 2 h while stirring activates the membrane fully to be used for sensor measurements. A number of membranes were prepared at different times but with same known consortia (mentioned above) immobilized on nylon membrane and studied for their reproducibility as well as for their shelf life. 2.4. Assembly of the microbial sensor and measurement of response The sensor response was measured by coupling the immobilized membrane to the cathode of the oxygen probe. A Clark type probe for dissolved oxygen was used as the physical transducer, which Table 1 Different concentrations of GGA S. no. 1 2 3 4
Concentration of GGA 1,920 3,840 7,680 12,000
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consisted of a platinum cathode as the working electrode and a silver anode as the reference electrode. The working and reference electrodes of the BOD sensor were connected to a potentiostat and the output current was recorded through a multimeter, which amplified the response to nA. An applied potential of −0.650 mV versus Ag/AgCl was delivered to the Pt-working electrode throughout all the measurements (Fig. 1). The BOD sensor measurements were made using steady state method. The steady state indicates the consumption of oxygen by microorganisms which diffuses from a sample solution to the membrane at equilibrium. In steady state method, the difference of current between the two steady states reflects the respiration rate of substrate and was used for BOD sensor estimation. The measuring time was 10–15 min followed by 15–16 min recovery time. In order to stabilize the BOD sensor system and measure the response of the immobilized microbial membrane, 100 ml of phosphate buffer (50 mM) pH 6.8 was placed in a thermo stated cell at 35 ± 2 ◦ C under constant and moderate stirring. Aliquots of stock synthetic/industrial samples were added after a stable current was attained for 30 min (initial steady state current). The current change (decrease) was observed after addition of the samples until a steady state was reached. The response was calculated on the basis of current difference between the initial steady state current and final steady state current. 2.5. Calibration Since GGA is used as a reference standard to standardize conventional 5-day BOD measurement, all the BOD measurements with the sensor in this study were carried out using these GGA solutions. A stock solution containing equal volume of 150 mg/l of glucose and glutamic acid with an assigned 5-day BOD value of 220 mg/l was prepared [1]. Different concentrations of GGA ranging from 5 to 120 mg/l were used for the calibration of the BOD by conventional method as well as by biosensor. 2.6. Wastewater samples Real wastewater samples, comprising both inlet as well as outlet samples, were procured over a period of time at different intervals, from a soft drink company of International repute, located near New Delhi. The pH value of the wastewater should be in the range of 6.6–8.5, whereas BOD should be around 30 mg/l before it is discharged outside. Wastewater characteristics of beverage wastewater included BOD in the range of 316 mg/l–2890 mg/l, and COD in the range of 605 mg/l–6778 mg/l. 2.7. Conventional COD and BOD 5-day test The COD and 5-day BOD tests of standard solution and all effluent samples were carried out according to the method described in Standard Methods for Examination of Water and Waste-waters [1]. 2.8. Measurement of BOD by biosensor The biosensor was used to estimate the BOD of a large number of beverage wastewater samples. The dilutions had to be calculated manually for each sample so that the BOD value falls within the range followed by the calculation of BOD. 2.9. Need for the development of software It was likely that by carrying out some more improvements and modifications, it may be possible to reach to a conclusion for the commercialization of the sensor for beverage industry. Some of the
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Fig. 1. Schematic diagram of the amperometric biosensor.
aspects that were considered for achieving the above were as follows: • Automated generation of the dilution required for the estimation of BOD. • DO correction factor for the instrument. 2.10. Software development Software, “BioSensBOD ” was developed on visual basics platform meant for graphical displays, BOD calculation, online data acquisition and other calculations. The developed software “BioSensBOD ”, which was developed on visual basic platform, has got two main parts: (A) In first part, users enter the parameters like sample description, membrane ID, % dilution, maximum time for taking readings, time interval between readings, and output file name in the form “parameters” shown in supplementary Fig. 1. Thereafter it jumps to the next form “Online Graph Plotting” (supplementary Fig. 2). This form has got four command buttons i.e. Start, Stop, Print and Exit. By pressing Start button, program starts acquiring data from the Keithley Multimeter (Model 2700) through RS232C interface and the date and time of starting of the acquisition of the data is displayed on the screen. The acquired data is also saved in the user defined output file. After the maximum time, the plotted graph can optionally be printed on the printer. (B) The second part is offline part. This has got further three parts: (i) membrane data, (ii) BOD calculator and (iii) offline graph plotting. (i) Membrane data: By selecting this user can enter the new membrane reference data at different GGA concentrations along with its ID, modify the existing membrane data, delete any membrane reference data or can also undelete the deleted membrane reference data. The reference membrane data form is shown in supplementary figure (Fig. 3). (ii) BOD calculator: This calculator asks the user to enter the membrane ID, I (i.e. Imax − Imin ) at 60GGA, I (i.e. Imax − Imin ) of the test sample, and the percentage dilution of the sample used. Thereafter, the program compares the present I at 60GGA with the reference I of the selected membrane and calculates the correction factor and applies it for the calculation of BOD level and the same is displayed in mg/l. In case the equivalent I for the sample reaches the saturation level, then program does the reverse calculation and gives the value of the dilution of the sample to
be used for proper measurements. The BOD calculator data is shown in supplementary Fig. 4. (iii) Offline graph plotting: This form asks the user to enter the file name whose data is to be plotted on the screen and then plots the data after reading the file. The user can also view the data of any selected file on the screen. The offline graph plotting form is shown in supplementary Fig. 5a and b. 2.11. BOD estimation of beverage wastewater using developed device The device was assembled by connecting the immobilized electrode to the developed highly stable constant volt source and multimeter which in turn is connected to a mobile PC through RS232C interface. Mobile PC is installed with developed software “BioSensBOD ” built on visual basics platform. This software acquires the real time current data from the multimeter and plots the same in the form of desired graphical display as well as save the same in the user defined file along with the time in seconds. In starting, electrode was dipped in sodium phosphate buffer placed on the magnetic stirrer and external polarization voltage was applied through the highly stable developed voltage source. Current through the electrode was measured by the multimeter. Then stability of the immobilized microbial membrane was observed as displayed by developed software installed in a mobile PC which in turn is connected to a multimeter through RS232C interface. Change in current (i.e. I = Imax − Imin ) was calibrated for different concentrations of GGA. Sample solution was replaced now with fresh sodium phosphate buffer and assembly was stabilized. In order to calculate the BOD, the percentage of the wastewater was added in sodium phosphate buffer, as determined by the software. Finally, BOD value of wastewater was calculated with the help of BOD calculator as mentioned above. This BOD value was compared to BOD5 as determined by titration-based method. 3. Results and discussion The BOD sensor was aimed at being highly capable for analyzing a sample of complex constituents with relatively low selectivity. The developed sensor responded to all kinds of biodegradable organic solutes present in the sample of beverage industrial wastewater. However, the problem remained as to how to determine the BOD load of any wastewater as and when it arrives in the laboratory, i.e. which dilution to use and whether the BOD values determined are
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Fig. 3. Graphical representation showing the comparative performance of biosensor with and without the use of software.
Fig. 2. Response curve of different concentrations of GGA as measured by the sensor: (a) membrane response, (b) linearity of membrane and (c) calibration curve.
comparable with the conventional method or not and how much time is required to determine the reliable BOD values. With this aim in mind, an extensive testing of beverage wastewater was undertaken by conventional method as well as by the developed BOD biosensor. 3.1. Calibration The calibration of the sensor involves correlating the sensor response with the 5-day BOD value of the solution. For calibration, the linearity in response is measured. The linear range of the sensor is defined as the substrate range that gives a signal directly proportional to the concentration. Linearity in response time over a certain concentration range is a measure of the detection capacity to analyze wastewater with varying concentrations. The BOD values of different concentrations of GGA as determined by the developed sensor showed linearity up to 60 mg/l of GGA, which corresponded to BOD values of 45 mg/l. The linearity of this range was satisfactory [R2 = 0.9914, Fig. 2(a–c)]. 3.2. Determination of BOD load of beverage wastewater with the developed software The COD and BOD of beverage wastewater varied from 1000 to 3400 and 300 to 2250 mg/l, respectively, over a period of time (supplementary Fig. 6). Based on the extensive data obtained
(Table 2), a software was designed which instantly gave the actual dilution of the wastewater to be used for the determination of BOD values. The correction factor was taken into consideration while integrating software with the device. Thus, it was possible to determine the BOD values of wastewater instantly after using just one concentration, thereby reducing the time (Fig. 3). The BOD values of inlet and outlet samples of effluent as determined by the developed software aided BOD biosensor are shown in Table 3. The results showed that the BOD values were comparable to BOD values determined by the conventional method and lay well within the range. In this case, COD was not required. Therefore, the determination of BOD load was accomplished in 90 min as against 5 days method without the need of performing COD analysis. The percentage variation in the BOD results was limited to ±8% in case of inlet samples and to ±10% in case of outlet samples. Variation was observed in the ratio of BODsensor :BOD5 in case of real wastewater .This was corroborated to the different measuring principles involved in both the cases as well as due to variability in composition of wastewaters. However, it was predicted by Liu and Mattiasson [9] that it is possible to minimize the problems by selecting suitable microbial strains as biological recognition elements. Rastogi et al. [14] reported that a BOD biosensor based on pre-tested, synergistic formulated microbial consortia was capable to sense the BOD load of a wide variety of industrial wastewaters having low moderate–high biodegradability. It has been emphasized that a pre-selection of microorganisms for their capability to degrade a range of pollutants can serve to develop an ideal BOD biosensor. Such a biosensor can cater to a variety of industrial effluents in determining the BOD load. Earlier, BOD biosensors have been tried for various synthetic substrates [8,10,11,14] but when applied to real wastewaters, they did not give good correlation with BOD5 values. 3.3. Repeatability and reproducibility As per the conventional BOD method, analysis of a GGA solution check with an average BOD5 value of 198 mg/l and a variability of precision of about ±15% is intended to be the reference for any laboratory. In the present study, the repeatability error of 3.49 and 4.45% was obtained in case of GGA (60 mg/l) when two membranes were used (Table 4). It has been reported earlier, the repeatability varies from ±2.4% to ±10% in case of single strain sensors with maximum value of ±10% in case of Pseudomonas putida, ±5% to ±11% for sensors based on the mixture of two identified strains with maximum ±11% in case of mixture of two identified bacteria, Citrobacter sp. and Enterobacter sp., ±1.3% to ±12.4% for those multi-strain based
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Table 2 Percentage deviation of BODs vs. BOD5: beverage wastewater without using software S. no.
COD (mg/l)
BOD5 (mg/l)
Sample percentage used
BOD sensor (mg/l)
Percentage deviation BODsensor vs. BOD5
1
2090
1270
2.75 3.00 3.25 3.50 3.75 7.00 10.00
1382 1256 1167 1086 1013 966 624
+8.82 −1.10 −8.11 −14.49 −20.24 −23.94 −50.87
2
2050
1250
2.75 3.00 3.25 3.50 3.75
1320 1282 1262 1232 1223
+5.6 +2.56 +0.96 −1.44 −2.16
3
1665
1232
3.00 3.25 3.50 3.75 4.00
1502 1456 1328 1245 1229
+21.92 +18.18 +7.79 +1.06 −0.24
4
1456
803
3.50 3.75 4.00 4.25 4.50 4.75
1156 1101 1093 1041 981 946
+43.96 +37.11 +36.11 +29.64 +22.17 +17.81
5
2702
1810
2.00 2.25 2.50 3.00 3.50 5.00 5.50
1960 1797 1752 1584 1486 724 673
+8.29 −0.72 −3.20 −12.49 −17.90 −60.00 −62.82
Table 3 Percentage deviation of BODs vs. BOD5: (a) beverage inlet wastewater and (b) beverage outlet wastewater S. no.
BOD5
BODs
Percentage deviation BODs vs. BOD5
BODs/BOD5
(a) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
610 460 790 810 630 520 660 729 826 1098 410 2250 300 777 1820 1245
605 449 783 878 626 543 673 710 848 1087 409 2105 295 785 1808 1275
−0.8 −2.4 −0.9 +8.4 −0.6 +4.4 +2.0 −2.6 +2.7 −1.0 −0.2 −6.4 1.7 +1.0 −0.6 +2.4
0.99 0.97 0.99 1.08 0.99 1.04 1.02 0.97 1.02 0.98 0.99 0.93 0.98 1.05 0.99 1.02
(b) 1 2 3 4 5 6 7 8 9 10
58 69 32 48 47 27 54 44 40 45
62 69 33 50 50 28 49 48 42 48
+6.9 −0.0 +3.1 +4.2 +6.4 +3.7 −9.2 +9.0 +5.0 +6.7
1.06 1.00 1.03 1.04 1.06 1.03 0.90 1.09 1.05 1.06
BOD5 estimation by 5-day BOD test BODs estimation by developed sensor.
sensors with maximum in case of heat killed cells and ±1.5% in case of predefined microbial consortia based sensors [8]. In the present study, in a sensor based on predefined, pre-tested microbial consortium, the reproducibility of 1.8% was obtained in case of GGA and of ±1.44% was obtained when real wastewater was applied for detecting the BOD load. Sakai et al. [18] reported the sensors based on magnetic activated sludge having reproducibility within ±5%. However, the determination of BOD of a wide variety of effluents and reproducibility in terms of monitoring of a wide variety of substrates remained a problem. 3.4. Operational stability Number of membranes were tested for operational stability in biosensor and it was found to be more than 400 days (supplementary Fig. 7) but the membrane was required to be conditioned after 120 days (data not shown). It has been reported [14] that a mixture of two identified bacteria gave an operational stability of 60 days with more than 200 measurements. The noncompliance of results with BOD5 was attributed to the presence Table 4 Repeatability and reproducibility of sensor membranes: repeatability of sensor with membrane AI–AVI (n = 12) using GGA S. no.
Membrane
Mean
S.D.
R.S.D.
Standard error
1 2 3 4 5 6
AI AII AIII AIV AV AVI
280.92 364.25 337.50 367.00 354.25 348.92
3.20 3.14 2.87 3.13 3.11 3.37
1.14 0.86 0.85 0.85 0.88 0.97
0.92 0.91 0.83 0.90 0.90 0.97
AI–AVI are same immobilized membranes prepared at different times with same known consortia.
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of many different microorganisms used in conventional method thus being able to biooxidize a larger range of organic substrates. The operational stability of a sensor is affected to a great extent upon the preconditioning of the membrane. The preconditioning has been defined in two ways one to increase the range of assimilation of substrates by the immobilized microbes [10,14], secondly to fully activate the immobilized microbes in a newly constructed membrane [13]. The activation of the membranes at various periods of time increases the operational stability. Li et al. [3] have reported that fully activated cells remained stable and sustained activity for more than 200 reproducible measurements over a period of two months. The operational stability of our developed sensor is 400 days which is the first report in this regard. 4. Conclusion An automated computer aided software integrated BOD biosensor was developed which is able to determine BOD load within one and half hour. The results of extensive testing of developed BOD biosensor on beverage wastewater over a period of time demonstrate that BOD values obtained are comparable to conventional BOD values, irrespective of varying load of wastewater (which might occur due to different operations occurring in industries). Moreover this shows good reproducibility and repeatability over a period of time. The membrane is stable up to 400 days with pre-conditioning at various intervals. This is the first report on the determination of BOD load of real wastewater, i.e. beverage industrial wastewater in matter of hours irrespective of BOD load at any given time. From above results we can conclude that the developed biosensor is pertinent for online, monitoring. Acknowledgements We acknowledge the Ministry of Environment and Forests, New Delhi, for financial assistance. We are also thankful to Prof. S.K. Brahmachari, Director, Institute of Genomics and Integrative Biology for providing necessary facilities. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.snb.2008.03.010.
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