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Rapid monitoring and assessment of pollutional load in

dairy waste water
Cite this: Anal. Methods, 2013, 5, 977

Purnima Dhall,a Rita Kumar,a T. O. Siddiqi,c Altaf Ahmedc and Anil Kumar*b

Received 25th October 2012
Accepted 10th December 2012
DOI: 10.1039/c2ay26269j
www.rsc.org/methods
In the present scenario, an attempt was made to develop an ideal device (BOD biosensor) with computer-
aided software capable of determining the varying BOD load of dairy waste waters and which facilitates
instant monitoring. The shelf life of the developed biosensor was more than 400 days. The results of
extensive testing of the developed BOD biosensor on dairy waste water over a period of time demonstrate
that the BOD values obtained by the device are statistically correlated with conventional BOD values,
irrespective of the varying load of waste water (which might occur as a result of different operations in
industry). The developed BOD biosensor shows good reproducibility and repeatability over a period of
time. Good correlation (r2 ¼ 0.991) was observed between the values obtained from the developed sensor
and conventionally estimated values. The repeatability of the measurements with the BOD biosensor dairy
industrial waste water samples (inlet and outlet) was within a percentage deviation of 10%.

Introduction examples, such as single cells,6–8 yeast cells,9,10 mixed cultures,11

High organic load discharge in freshwater bodies is an intricate
problem faced by government organizations. Discharging
standards have been given to industries, but the monitoring of
organic load through conventional methods has some draw-
backs which makes this slow work for those in industry. In case
of dairy industries, India's is the largest producer of dairy based
wastewater in the world.1 The characteristics of wastewater vary
from industry to industry depending on the manufacturing
process and the products being manufactured.2,3 Dairy waste-
water rich in biodegradable organics and nutrients contains
fatty acids, lactose, proteins and salts, as well as the chemicals
used during cleaning processes. Effluent varies in pH from 4.2
to 9.4 with an increased concentration of suspended solids (0.4–
2 g L1).1,2,4,5 For proper treatment of wastewater, regular
monitoring is required and if not treated, the wastewater causes
water pollution which may lead to eutrophication. The need for
a fast, portable and cost effective method for environmental
monitoring has stimulated the development of a variety of eld
analytical tools such as biosensors. A biosensor is a device that
transduces a selective biochemical response into a measurable
signal. Several biosensor methods for biochemical oxygen
demand (BOD) measurement have been developed. Some
a
Institute of Genomics and Integrative Biology, Mall Road, Delhi-110007, India.
E-mail: purnimadh@igib.in; rita@igib.res.in; Fax: +91-11-27667471; Tel: +91-11-
27662133 ext. 154
b
National Institute of Immunology, Aruna Asif Ali Marg, New Delhi-110067, India.
E-mail: anilk@nii.ac.in; Fax: +91-11-26742125; Tel: +91-11-26717106, 26703525
c
Jamia Hamdard University, Hamdard Nagar, New Delhi-110062, India. E-mail:
tariq117@rediffmail.com; aahmed@jamiahamdard.ac.in; Fax: +91-11-26059663;
Tel: +91-11-26059688 ext. 5535, 5540
microbial fuel cell biosensors,12–19 and whole cell biosensors20–23
have been developed since Karube et al. rst reported a BOD
biosensor based on a Clark dissolved oxygen electrode immo-
bilized microorganism in 1977.24 Major drawbacks associated
with existing sensors are a low shelf life, non reproducibility of
the activity of the immobilized membrane and the long time
taken to complete the reaction.
In the present scenario, an attempt was made to develop an
ideal device (BOD biosensor) with computer-aided soware
capable of determining the varying BOD load of dairy waste
waters and which facilitates instant monitoring. This would
help users to determine the efficiency of treatment systems and
permit instant action to improve this efficiency.
Material and methods
Chemicals
Charged nylon membrane (Sigma) with a pore size of 0.45 mm
was used throughout the investigation. D-Glucose and D-gluta-
mic acid were obtained from Sigma, Germany. The other
chemicals used to prepare the growth medium were procured
from Hi-Media, India.
Microorganisms and culture conditions
The same procedure was followed as mentioned in Rastogi
et al.25 The inoculum was prepared by inoculating one loopful of
individual bacterial isolates, in 50 mL Erlenmeyer asks of
sterilized nutrient broth. The inoculated broths were incubated
in an orbital shaker at 37  C for 16 h so as to obtain actively
growing mother cultures. The microorganisms were main-
tained at 4  C in the same medium prepared with 2% agar.

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Analytical Methods
Preparation of the cell suspension
The cells were harvested at log phases of growth from the broth
culture by centrifugation at 7000 rpm for 30 min at 4  C. Cells
were washed twice with 50 mM phosphate buffer, pH 6.8. The
resulting cell pellet was suspended in 50 mM phosphate buffer,
pH 6.8 and the cell slurry was stored at 4  C until use.
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Immobilization of the bacteria
The cell slurry was immobilized on the nylon membrane by
ltering small aliquots under moderate vacuum. The immobi-
lized microbial membrane thus prepared was le to dry for
18–20 hours at room temperature. Finally, the dried
membranes were transferred to sodium phosphate buffer
solution and stored at 4  C until further use.
Assembly of the microbial sensor
The response was measured by coupling the immobilized
membrane to the cathode of the oxygen probe. Nylon net (400
mesh) was attached to the immobilized microbial membrane, and
two of these were xed on the surface of the working and reference
electrodes by means of a rubber O-ring. The working and reference
electrodes of the BOD sensor were connected to a potentiostat and
the output current was recorded through a Keithley multimeter. A
highly stable potential of 0.650 V was applied to the Pt-working
electrode throughout all the measurements.
Conditioning of the membranes in different concentrations of
GGA (glucose–glutamic acid)
Conditioning of the immobilized microbial membrane was
required before its use. The membranes were preconditioned in
different concentrations of GGA. The response of the bacteria
was calculated (DI) and observed for 60GGA.
Preparation of a response curve, linearity curve and
calibration curve for the immobilized microbial membrane
GGA was used as a reference standard for all BOD measure-
ments of the samples as well as for the calibration of the
biosensor. Stock solution of GGA with a concentration of
12 000 mg L1 was prepared. Different aliquots from the stock
solution of GGA were added in the measuring cell of the BOD
biosensor system, to achieve the desired GGA concentration of
15–300 mg L1 (having a BOD of 11–220 mg L1). The response
of the biosensor with different concentrations of GGA was
observed and recorded. The linearity curve was plotted with the
values obtained for the response curve. For the calibration curve
the readings were plotted and, on the same graph, a second
mantissa was drawn showing the conventional BOD (BOD5)
values against the same GGA concentrations as used with the
developed BOD biosensor.
Wastewater samples collection and characteristics
Real wastewater samples, comprising both inlet as well as outlet
samples, were procured from the dairy industry located near
New Delhi over a period of time. The raw effluent sampling
978 | Anal. Methods, 2013, 5, 977–981
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point was aer the primary screening and sedimentation but
before the aeration tank, and the outlet wastewater aer treat-
ment was collected. The samples were stored at 4  C. Charac-
teristics of dairy wastewater included BOD in the range of 923–
3670 mg L1 and COD (chemical oxygen demand) in the range
of 1387–5320 mg L1 for the inlet, and in the case of the outlet
BOD and COD vary in the range of 8–53 mg L1 and 52–218 mg
L1 respectively.
Conventional BOD 5 day test
The 5 day BOD test of the standard solution and the dairy
effluent inlet and outlet samples were carried out according to
the method described in Standard Methods for the Examination
of Water and Wastewaters.26
BOD estimation of dairy waste water using the developed
device
The device was assembled by connecting the immobilized
membrane and nylon cloth to electrodes which were connected
to a multimeter and in turn connected to a mobile PC through an
RS232 interface. The mobile PC was installed with the developed
soware “BiosensBOD,” built on the visual basic platform. To
start with, the electrode was dipped in sodium phosphate buffer
and an external polarization voltage was applied from the highly
stable developed voltage source. Then the stability of the
immobilized microbial membrane was observed. The response
was observed in terms of the change in current (i.e. DI ¼ Imax 
Imin) for 60GGA. The sample solution was replaced with fresh
buffer and the assembly was stabilized. An amount of dairy
wastewater was added and the BOD was calculated with the help
of soware. This BOD value was compared to the BOD5 value as
determined by conventional methods.
Reproducibility of the immobilized microbial membrane
To check the reproducibility of the immobilized membrane, a
GGA concentration of 60 mg L1 was used and the response of
the membrane was checked as mentioned above.
Precision
Precision in the actual sense means repeatability and repro-
ducibility. Repeatability is the standard deviation of a tested
single sensor by one operator under the same conditions while
reproducibility is the standard deviation of a series of tested
sensors by more than one operator. However the terms are
relative with respect to the parameters. The obtained results were
compared with the values estimated by conventional methods.
Results and discussion
Response curve, linearity curve and calibration curve
Before testing the samples, the BOD sensor was used to analyze
the BOD values of different concentrations of the standard GGA
solution, the BOD5 measurements for which were simultaneously
carried out. The membrane response was checked by using a
different concentration of GGA (Fig. 1). For plotting the linearity
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Fig. 1 The response curve using different concentrations of GGA (5–75 mg L1).

curve, the response of the membrane was measured. The linear
range of the sensor is dened as the substrate range that gives a
signal directly proportional to the concentration.27,28 The linearity
curve for the immobilized membrane was also plotted and the
value of r2 is equal to 0.99 (Fig. 2). The calibration of the sensor
involves correlating the sensor response with the 5 day BOD value
of the solution (Fig. 3).29
BOD estimation using the developed sensor system
The BOD sensor was used to estimate the BOD of dairy
wastewater samples. For the same samples, the 5 day BOD was
also determined by the conventional method. The total
analyzing time was approximately 15 min. Means and standard
deviations of the results from the two methods were examined
using Microso excel soware (Table 1).

Fig. 2 The linearity curve.

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Analytical Methods Paper

Table 3 Percentage deviation of BODs vs. BOD5: (a) dairy inlet wastewater and
(b) dairy outlet wastewater

Percentage deviation
Sample no. BOD5 BODs BODs/BOD5 BODs vs. BOD5

(a)
1 3670 3605 1.0 1.8
2 1880 1891 1.0 0.6
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3 1612 1523 0.9 5.5

4 1392 1456 1.0 4.6
5 1315 1419 1.1 7.9
6 1109 1058 1.0 4.6
7 1050 1075 1.0 2.4
8 971 952 1.0 2.0
9 923 960 1.0 4.0
10 900 904 1.0 0.0

(b)
1 53 49 0.9 7.5
2 52 50 1.0 3.8
3 49 52 1.1 6.1
4 47 45 1.0 4.3
Fig. 3 The calibration curve. 5 39 39 1.0 0.0
6 36 38 1.1 5.6
7 34 36 1.1 5.9
Table 1 Comparison of BOD sensor values with BOD5 values 8 29 29 1.0 0.0
9 27 28 1.0 3.7
BOD Percentage 10 10 11 1.1 10.0
Statistical BOD5 biosensor deviation
parameters (mg L1)000 (mg L1) w.r.t BOD5
Merlin et al.,30 developed semi-specic microbial biochemical
Average (n ¼ 20) 1533 1532 +0.06
Median 1446 1336 +7.6 oxygen demand (BOD) biosensors using living culture immo-
Correlation coefficient b/w BOD5 and BOD biosensor 0.97 bilized using agarose. Their results show that the service life
was 60 days for E. coli and P. uorescens based biosensors and
40 days for R. terrigena based biosensors, whereas in the shelf
life was up to 400 days in case of a soware incorporated
Reproducibility of the immobilized microbial membrane biosensor.30
It should be noted that the biological recognition element was
checked before each sample analysis with GGA to check the
activity and reproducibility of the immobilized consortia. The Conclusion
standard deviation comes out to be 1.16, and shows a relative An automated computer aided soware integrated BOD
standard deviation of 0.33 with a standard error of up to 1.16, so biosensor was developed which is able to determine the BOD
the system gives stable measurements with good repeatability load in 15 min. The results of extensive testing of the developed
(Table 2). BOD biosensor on dairy wastewater over a period of time
demonstrate that the BOD values obtained are comparable to
conventional BOD values, irrespective of the varying load of
Reproducibility of the sensor wastewater (which might occur as a result of different opera-
Extensive testing of wastewater samples was done to test the tions in industry). Moreover this biosensor shows good repro-
reproducibility of the sensor. The results obtained were ducibility and repeatability over a period of time. Good
compared with the results from conventional methods correlation (r2 ¼ 0.991) was observed between the values
(Table 3). The reproducibility results show the percentage obtained from the developed sensor and conventionally esti-
deviation to be within the range of 7% for the inlet and 10% mated values.
for the outlet.

Acknowledgements
Table 2 Reproducibility of the immobilized microbial membrane using 60 mg We are thankful to Prof. S. K. Brahmachari, DG, CSIR for
L1 GGA
providing the necessary facilities. The authors also acknowledge
Percentage Mean SD RSD Std error the members of the dairy industry, for extending their cooper-
ation for providing wastewater samples, whenever required and
60 mg L1 GGA (n ¼ 20) 350 1.16 0.33 1.16 the generous hospitality offered to us upon each visit.

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Paper
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