BOD analysis of industrial effluents 5 days to 5 min

BOD analysis of industrial effluents: 5 days to 5 min
Shikha Rastogi
a
, Pratima Rathee
a
, T.K. Saxena
b
, N.K. Mehra
c
, Rita Kumar
a,
*
a
Centre for Biochemical Technology, Delhi University Campus, Mall Road, Delhi 110 007, India
b
National Physical Laboratory, Dr. K.S.Krishnan Marg, New Delhi 110 012, India
c
Department of Zoology, Delhi University, Delhi 110 007, India
Abstract
Wastewater generation and its subsequent treatment is a major problem for every industry and for the society as well. Prior to
treatment, the wastewaters need to be monitored so as to permit their discharge into the local water resources. Amongst all the
parameters for which the wastewaters are monitored, biochemical oxygen demand (BOD), is one of the most important and fre-
quently used parameters for estimating the level of water pollution. The control of wastewater treatment plants is very difficult or
even impossible using the classical determination method for BOD because of its high time consumption (3–5 days). The need for
fast, portable and cost-effective methods for environmental monitoring has stimulated the production of a variety of field analytical
tools such as biosensors. Biosensors are device that have several unique features such as compact size, simple to use, one step re-
agentless analysis, low cost and quick-real time results. The conventional BOD measurement requires 3–5 days, which a microbial
BOD biosensor senses within minutes. A number of microbial BOD sensors have been developed nationally and internationally. The
drawback of such developed sensors is that they cannot be used for all types of industrial and domestic wastewaters.
Our developed BOD biosensor is based on a pre-tested, synergistic formulated microbial consortium. It is capable to sense the
BOD load of a wide variety of synthetic as well as industrial wastewaters having low–moderate–high biodegradability within

minutes. The sensor BOD values show a good linear relationship with the BOD values obtained using the conventional method upto
a GGA concentration of 90 mg/l (r ¼ 0:938). BOD values of real wastewater samples from different industries viz, distillery dairy
and tannery were analysed using the developed sensor. The BOD sensor results were found to be comparable with those obtained
using the conventional 3-day method.
Ó 2002 Elsevier Science B.V. All rights reserved.
PACS: 82.47.Rs
Keywords: BOD biosensor; Immobilized microbial membrane; Biochemical oxygen demand; Microbial consortium; Industrial wastewaters
1. Introduction
The biochemical oxygen demand (BOD) test is a
crucial environmental index to determine the relative
oxygen requirements of wastewaters, effluents and pol-
luted waters. It measures the molecular oxygen utilized
during a specified incubation period for the biochemical
degradation of organic material (carbonaceous demand)
and the oxygen used to oxidize inorganic material such
as sulphides and ferrous ions. It can also be a measure of
oxygen used to oxidize reduced forms of nitrogen
(nitrogenous demand), unless their oxidation is pre-
vented by an inhibitor [1]. The conventional BOD test
requires a five day incubation period at 20 ° C and de-
mands skill in determination, thereby, making it un-
suitable for process control. Thus, it is necessary to
develop a measurement method that could circumvent
the weaknesses of the conventional method. The fast,
portable and cost effective methods for environmental
monitoring has stimulated the development of a variety
of field analytical tools such as biosensors. Biosensors
are devices that transduce a selective biochemical re-
sponse to a measurable signal. Several biosensor meth-
ods for BOD measurement have been developed.

The first report of BOD biosensor was published by
Karube et al. in 1977. After that, several kinds of mi-
crobial BOD sensors have been developed and various
modifications have been carried out [4,6,8,9,12,15,16,
18–29]. Most of the above reported BOD sensors con-
sisted of a synthetic membrane with single or a random
*
Corresponding author. Tel.: +91-11-7666156/157/7602; fax: +91-
11-7667471.
E-mail address: (R. Kumar).
1567-1739/02/$ - see front matter Ó 2002 Elsevier Science B.V. All rights reserved.
doi:10.1016/S1567-1739(02)00199-2
Current Applied Physics 3 (2003) 191–194
www.elsevier.com/locate/cap
combination of immobilized microorganisms serving as
biocatalyst. A rapid and reliable BOD sensor should aim
at being highly capable of analysing a sample of com-
plex constituents with relatively low selectivity. Thus the
sensor can respond to all kinds of biodegradable organic
solutes in the samples. It is also important that the
sensor should give results comparable to those obtained
using the conventional BOD method. Design and de-
velopment of a BOD sensor, based on a pre-tested for-
mulated, synergistic microorganism in combination with
an oxygen electrode, was therefore considered in the
present course of study.
The developed BOD biosensor is capable of assimi-
lating most of the organic matter present in different
types of wastewaters as well as industrial effluents. The
aim of present study was to obtain good agreement

between results of the sensor BOD measurement and
those obtained from the conventional BOD analysis.
2. Materials and methods
A formulated, synergistic and pre-tested microbial
consortium used as a reference seeding material for
BOD analysis [10,14] was incorporated as the biocom-
ponent in the developed BOD sensor. The microorgan-
isms comprising the microbial consortium were
harvested in their log phase of growth from the respec-
tive broth cultures. The cell pellet was suspended in 50
mM phosphate buffer solution (pH 6.8) to obtain
the cell slurry. This cell slurry was immobilized on
charged nylon membrane by filtering small aliquots
under moderate vacuum. The immobilized microbial
membrane prepared in the above said manner was left to
dry for 18–20 h at room temperature. Finally, the dried
membranes were transferred in buffer solution and kept
at 4 °C, till further use. The immobilized microbial
membrane was then coupled to the electrode by holding
the membrane against the teflon gas permeable mem-
brane by means of a nylon net to get a complete elec-
trode assembly. The response of the electrode was
measured in term of current (nA) obtained on a mul-
timeter.
In a few minutes after the electrode assembly was
immersed into a buffer solution, (50 mM K
2
HPO
4
–

KH
2
PO
4
buffer, pH 6.8) the current become constant
because the diffusion rate of oxygen into the microbial
film from the bulk of the solution reaches equilibrium
with the consumption rate of oxygen by endogenous
respiration of the immobilized microbes. This current
level is named as Ôinitial basal currentÕ. When appro-
priate aliquots from a standard BOD solution were
added into the stabilized electrode assembly, the current
level decreased as the biodegradable compound diffuse
into the microbial film from the bulk of the solution.
Then, in few minutes, the current of the dissolved oxy-
gen (DO) probe reached another constant current level
known as the Ôfinal basal currentÕ. The difference be-
tween the initial and final basal current values was de-
fined as change in current (DI). Because the magnitude
of the DI is proportional to a concentration of imme-
diately biodegradable organic compounds in a sample in
a certain range, an unknown BOD concentration in a
sample is predictable based on the magnitude of DI
observed.
The developed BOD sensor was calibrated using a
standard glucose–glutonic acid (GGA) solution [1].
BOD of the industrial effluents were measured using the
BOD sensor and BOD
5
test based on the dilution

method described in JISK0102 [5] and standard methods
[1]. The results obtained using the two measurement
systems were compared.
3. Results and discussion
The overall characteristics of a BOD biosensor are
determined by the characteristics of the microbial
membranes used in combination with the electrochemi-
cal sensor [3]. First amongst them is the effect of cell
population on BOD values. A number of BOD biosen-
sors have been developed using single organism or a
random combination of organisms, but mixed cultures
are shown to be an efficient biodegrading agent for
organic compounds in aqueous solution, with good ki-
netics, sensitivity, stability and reproducibility [7]. Keep-
ing this in view, a formulated microbial consortium,
developed and extensively tested with a wide range of
synthetic as well as industrial wastewaters was used as
biocatalyst for the construction of the BOD biosensors
in the present study [11,14].
The BOD values as observed by the developed BOD
biosensor reflect the concentrations of the dissolved
organic substances which are assimilated/metabolized
by the immobilized microbes [17]. The assimilation of
organic substances in turn depends on the metabolic and
physiological state of the immobilized biocatalyst, i.e.,
the microorganisms in use, their type, their phase of
growth and density on the support. The prepared im-
mobilized microbial membrane was therefore charac-
terized w.r.t. the above stated parameters. The findings
are depicted in Table 1.

The developed and characterized BOD sensor was
used to analyze the BOD values of different concentra-
tions of the standard GGA solution, the BOD
5
of which
were simultaneously carried out. These values were
utilized to plot a calibration curve as illustrated in Fig.
1. A linear relationship was observed between the cur-
rent difference (between initial steady state current and
final steady state current) and the 5-day BOD of the
standard solution upto a concentration of 90 mg l
À1
.
The linear range of the sensor is defined as the substrate
192 S. Rastogi et al. / Current Applied Physics 3 (2003) 191–194
range that gives a signal directly proportional to the
concentration [2]. The lower limit of detection was 1
mg l
À1
BOD, by the developed sensor. The current was
reproducible within Æ5% of the mean in a series of 10
samples having 44 mg l
À1
BOD, using standard GGA
solution.
The developed BOD sensor was used to estimate the
BOD of real wastewater samples. For the same samples,
5-day BOD was also determined by the conventional
method for comparison with BOD values estimated by
the sensor. The wastewater samples analyzed were those

emanated from dairy, distillery and tannery industries.
Each of these wastewaters were diluted appropriately
with buffer depending on their BOD load. Table 2 shows
the comparative BOD values for different industrial
wastewater as examined by both the methods. While
estimating the BOD values with the sensor, different
dilutions of the same wastewater showed varied results.
This is so because the change in composition of a
wastewater sample due to dilution could apparently in-
fluence the bacterial respiration rate [13]. Moreover, the
high molecular weight substances that are impervious to
the immobilized microbial membrane may remain un-
noticed while estimating the BOD with the help of the
sensor.
In addition, the immobilized bacteria might assimi-
lates various organic substances in distinct metabolic
pathways or procedures, resulting in different levels of
oxygen consumption. If wastewater samples with differ-
ent composition are analyzed, the immobilized bacteria
could show different respiration rates even although the
samples have the same BOD
5
values. Moreover, there
might not be a universal standard solution that would be
suitable for the calibration of real wastewater samples of
different compositions.
Acknowledgements
We acknowledge the Ministry of Environment and
Forests, New Delhi for financial assistance. The author
Shikha Rastogi greatfully acknowledge the CSIR, New

Delhi for Senior Research fellowship.
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