Environmental Technology

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Bioremediation of greywater using a novel

bacterial–fungal consortium: optimization and
validation of the operating parameters in vitro

Nikita Rajpal , Jatinder K. Ratan , Neetu Divya & Ananda Vardhan Hebbani

To cite this article: Nikita Rajpal , Jatinder K. Ratan , Neetu Divya & Ananda Vardhan
Hebbani (2021): Bioremediation of greywater using a novel bacterial–fungal consortium:
optimization and validation of the operating parameters in�vitro , Environmental Technology, DOI:
10.1080/09593330.2021.1882582

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Published online: 11 Feb 2021.

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ENVIRONMENTAL TECHNOLOGY
https://doi.org/10.1080/09593330.2021.1882582

Bioremediation of greywater using a novel bacterial–fungal consortium:

optimization and validation of the operating parameters in vitro
Nikita Rajpala, Jatinder K. Ratana, Neetu Divyaa and Ananda Vardhan Hebbani b

a
Department of Chemical Engineering, Dr. B.R. Ambedkar National Institute of Technology, Jalandhar, India; bDepartment of Biotechnology,
New Horizon College of Engineering, Bangalore, India

ABSTRACT ARTICLE HISTORY

In the present study, removal of pollutants in greywater was investigated using a novel bacterial– Received 7 May 2020
fungal consortium. Response surface methodology was used for the optimization of process Accepted 22 January 2021
variables like pH, temperature, inoculum size, and Carbon/Nitrogen (C/N ratio) for degradation
KEYWORDS
of pollutants. Experiments were based on Box Behnken statistical design and the results show a Greywater; COD; RSM;
good fit with the quadratic model, coefficient of determination (R 2) value of 0.9499. The sulphate; optimization
reliability of the model was established by various statistical parameters like lack of fit, pure
error, and residual sum of squares. The optimized conditions for maximum reduction in
chemical oxygen demand, oil & grease and sulphate were found to be 78.7%, 82.6% and 89.7%,
respectively after 96 h of incubation of the reaction mixture at pH 7; temperature 35°C;
inoculum size 150 µl and C/N ratio of 1:2. Our results clearly demonstrate that the developed
novel bacterial–fungal consortium can be a cost-effective, safe, and environment-friendly
alternative for remediation of greywater.

1. Introduction
Water supplied to the developed communities globally
is of 100% potable quality [1]. However, only a mere
3% of the supplied water is understood to be used up
for drinking and cooking purposes, while the remaining
major portion referred to as ‘greywater’ (∼ 65–75%) is
used for domestic activities such as bathtubs, showers,
hand basins, laundry machines and cleaning purposes
[2]. Even though the precise composition of greywater
depends on its source from where the water is drawn
[3], it generally does not include the wastewater that is
generated from toilet use, which is referred to as ‘black
water’ [4]. Composition of greywater (i.e. water from
bathroom, laundry or kitchen sinks) is known to be
greatly influenced by water quality in the area of
sample collection [5]. A variety of contaminants, includ-
ing acidic and alkaline substances, suspended/dissolved
solids, fats, oil/grease, heavy metals and synthetic
chemicals, are likely to be present in greywater [6,7].
Greywater is considered the most preferred waste-
water resource for treatment and reuse because of its
minimal levels of contamination, thus making it a rela-
tively easier resource to treat in comparison to the
other forms of wastewaters [8,9]. Among the different
treatment methods for greywater, bioremediation tech-
nology has been proved to be a very effective method
by many researchers over the last few years [10–13].
Nutrients and organic compounds present in greywater
make it a favoured substrate for the growth of microor-
ganisms and research has been carried out earlier,
wherein greywater was treated both under aerobic
[14] and anaerobic conditions [15], using different bac-
terial, fungal and algal species [16]. Different bacterial
strains have been proved to have evolved specific

CONTACT Ananda Vardhan Hebbani h.anandavardhan@gmail.com

© 2021 Informa UK Limited, trading as Taylor & Francis Group
2 N. RAJPAL ET AL.

enzyme systems that can degrade pollutants under assist the parameters related to the described novel con-
aerobic and anoxic conditions, thereby having the sortium for greywater treatment.
potential for bioremediation [17]. Similarly fungi and Highlighting on the importance of the BBD approach,
algae are also known to achieve pollutant reduction by this present study was designed to investigate the inter-
multiple mechanisms wherein the complex organic/ active effects between each operational condition (pH;
inorganic contaminants are degraded into cellular con- temperature; inoculum size and C/N ratio) and pollutant
stituents such as lipids and carbohydrates [18]. reduction process. Moreover, greywater sample was
Reports claim that the bioremediation of greywater analysed before and after treatment for various par-
has been achieved using different pure microbial cul- ameters using novel consortium.
tures under in vitro conditions, wherein pure single
microbial cultures were used to remediate either a
single pollutant or a combination of the polluting 2. Materials and methods
factors present in the greywater [19–22]. However, 2.1. Chemicals
many of the earlier reports clearly acclaim the poorer
performance of pure cultures towards mixture of pollu- All chemicals required for the present study were
tants [20,23]. In this context, the current work is being obtained from Sigma Aldrich. Solvents were purchased
designed to look at the greywater bio-remediating from Hi-media Laboratories Pvt. Ltd., Mumbai, India. All
capacity of microbial consortia since the combination other chemicals used were of the highest purity avail-
of microbial cultures would have a better pollutant able and of analytical grade.
stress withstanding capacity, due to their symbiotic
inter-relationship which would function synergistically
to exhibit the bio-remediating capacity [20]. Moreover, 2.2. Greywater sampling and its characterization
a novel microbial-fungal consortium has never been Greywater sample (excluding toilet waste) utilized in this
used to treat domestic greywater, which is being study was manually collected from household drains of
researched upon and optimized and validated in the apartment in Whitefield (between 12° 58′ 17.0004′′ N,
current work. 77° 45′ 0.4680′′ E), Bengaluru. Raw greywater was col-
Implementation of Response Surface Methodology lected using acid-washed sample bottles and immedi-
(RSM) for design optimization is a best and effective ately transported to the laboratory and kept in reserve
alternative for decreasing the cost of expensive analysis for further study in a refrigerator below 4°C to avoid
methods [24]. For empirical model building, RSM is a col- any physicochemical changes. Initially, suspended
lection of mathematical and statistical techniques. It is solids and other impurities in greywater sample used
an apt tool for finding the most suitable condition for in this study were filtered using Whatman filter paper
multiple factor experiments, search for relationship grade 1 made up of nylon membrane with a 0.45-µm
between factors, and prediction of response [25]. By suit- pore size. Before the experiment, some raw character-
able designing of experiments, optimization of a istics of greywater sample have been tested as stated
response, which is influenced by various independent by the standard method for the examination of water
variables (factors), can be performed. The resultant and wastewater [29], depicted in Table 1. It includes
model is a full illustration of the independent variables,
their interactions and their influence on the responses.
Table 1. Characterization of greywater.
A comparison between the Box–Behnken Design (BBD)
Greywater without any Greywater with
and other response surface designs like central compo- Characteristics treatment treatment
site design (CCD) has revealed the fact that the BBD pH 6.2 7.26
matrix is slightly more coherent than CCD and much Colour Milky Clear
Odour Pungent Odourless
more efficient than the three-level full factorial designs Turbidity 40.0 NTU 1.30 NTU
[26]. The BBD approach saves cost and time with its Electrical 2048.17 μS/cm 480.70 μS/cm
conductivity
great accuracy on describing effects of the interaction Total suspended 680.67 mg L−1 136.76 mg L−1
between different independent factors and predicted solids
responses. On a broader scale, it works best for overall Total dissolved 1230 mg L−1 928.34 mg L−1
solids
optimization process in terms of expenditure and Total solids 1910.43 mg L−1 504.6 mg L−1
keeping away experiments done under extreme con- BOD 540.43 mg L−1 85.90 mg L−1
TOC 385.90 mg L−1 96.23 mg L−1
ditions [27,28]. This would imitate the real process COD 1165.6 mg L−1 249.89 mg L−1
execution under field conditions. However, little con- Oil and grease 58.0ppm 10.34 mg L−1
Sulphates 95.6 mg L−1 10.34 mg L−1
sideration has been given to the BBD approach to

pH, colour, odour, turbidity, Electrical conductivity (EC),
total suspended solids, total dissolved solids, chemical
oxygen demand (COD), Biochemical oxygen demand
(BOD), total organic carbon (TOC), oil & grease, sulphate
and heavy metals (Fe and Mn).
The pH was measured with the help of a pH meter,
while EC and TDS were determined by a meter (Eutech
con 700). The turbidity was measured by the Nephelo-
metric method. Total solids (TS) and oil & grease (O&G)
were estimated by the gravimetric method. Total sus-
pended solids (TSS) were determined by subtracting
total dissolved solids (TDS) from total solids (TS). BOD
was estimated by preparing a required volume of
dilution water with the addition of nutrients and incu-
bation period of five days at 25°C, while chemical
oxygen demand (COD) determination was based on
rapid dichromate oxidation method. Total organic
carbon (TOC) was measured using TOC analyser (Shi-
madzu). Inductively coupled plasma mass spectrometry
(ICP-MS) was used to detect heavy metals, Manganese
(Mn) and Iron (Fe).
2.3. Micro-organisms and stock cultures
Freeze-dried microbial isolates Micrococcus luteus
(MTCC 4698), Rhodococcus equi (MTCC 6939) and
fungal isolate Aspergillus niger (MTCC 16888) used in
this study were purchased from The Institute of
Microbial Technology (IMTECH) Chandigarh, Punjab,
India. The cultures were revived according to American
Public Health Association [29] protocols. Stock cultures
were refrigerated at 4°C in the refrigerator. Furthermore,
for degradation studies of pollutants in greywater fresh
cultures from stock were prepared. The above selected
bacterial and fungal strains were selected on the basis
of previous reports suggested for their degradation
potential [30–34].
2.4. Culture maintenances, inoculum preparation
and isolation of micro-organisms
The cultures were maintained on nutrient agar and
potato dextrose agar (PDA) slants, respectively at 4°C
and preserved for further study. The stored cultures
(M. luteus (MTCC 4698), R. equi (MTCC 6939) and fungal
isolate A. niger (MTCC 16888)) were inoculated on nutri-
ent agar and potato dextrose agar plates, respectively
for growth. The inoculated medium with cultures was
statically incubated for 48 h at 32°C and 96 h at 37°C
for bacteria and fungus, respectively. From each plate,
suspension cultures were also prepared in 100 ml of
autoclaved inoculum media. The flasks, containing indi-
vidual cultures, were kept on a shaker incubator
ENVIRONMENTAL TECHNOLOGY 3
(150 rpm) for 48 h at 32°C for bacteria, and 96 h at 37°
C for fungus.
2.5. Assembling of consortium and bio-
interaction studies
Bacterial–fungal consortium was prepared aseptically
transferring the equal volumes (50 mL) of 48 h
M. luteus, R. equi and 96 h grown A. niger strain into a
Erlenmeyer flask containing soybean casein agar (SCA)
medium, followed by 96 h incubation at 32°C. Conse-
quently, the consortium obtained was used as a source
of inoculum for further studies. To check the antagonism
between bacterial and fungal consortium, petriplate
bioassay was performed in which the above-mentioned
strains were grown on a single petriplate containing
composite broth medium (CBM) and incubated at 28°C
for five days [35].
2.6. Experimental design and optimization study
The culture conditions were examined by implementing
RSM to interpret the effect of important variables on
degradation. Based on one factor at a time experiment,
the four significant factors selected as the independent
variables were pH, Temperature (°C), Inoculum size (µL)
and C/N ratio were studied and modelled for consor-
tium. A four-level Box – Behnken factorial design, con-
sisting of 30 experimental runs in one block with three
replicates at the centre point, was generated by the
Design – Expert software (Stat-ease, trial Version
10.0.6.) This method is suitable for fitting a quadratic
surface and it helps to optimize the effective variables
with a minimum number of experiments as well as to
analyse the interaction between the variables.
This design requires an experimental number of runs
according to
N = R4 + R3 + R2 + R + CP (1)
where R is the factor number which is 4 in this case and
Cp is the number of replications at the centre point
which has been set at 5 in the present study.
The coded values (Table 2) of the process parameters
are determined by the following equation
Xo
Xc = Xa − (2)
DXf
where Xc and Xa are the coded and actual values of the
independent variables, respectively. Xo is the actual
value of the independent variable at the centre point,
and Xf is the step change of Xa.
The analysis of experimental data was performed to
estimate the response of the dependent variable fitted
4 N. RAJPAL ET AL.
Table 2. Level of different process variables in coded and un-
coded forms for degradation of pollutants in greywater.
Factor range and levels(coded)
Code Name of Variables −1 0 +1
A pH 6 7
B Temperature (oC) 25 30
C Inoculum size (µL) 100 150
D C/N ratio 1:1 1:2
into a second-order polynomial.
Y = bo + b1A + b2B + b3C + b4 D + b12AB
+ b13 AC + b14AD + b23 AC + b24AD
+ b11A2 + b22B2 + b33C2 + b44D2
where Y = Predicted response, A,B,C,D are independent
variables, b0 is the offset term, b1, b2, b3, b4 are linear
effects, b11, b22, b33, b44, are squared terms and b12,
b13, b14, b23, b24 are interaction terms.
Statistical significance of the model equation and
model term was evaluated by Fisher’s test and Analysis
of variance (ANOVA). Quality of quadratic model
equation was expressed by the determination of coeffi-
cients R 2 and adjusted R 2. Optimal values are obtained
by solving the regression equation and three-dimen-
sional (3D) response surfaces were plotted by varying
two variable levels while keeping the other variables at
a constant level to directly exhibiting the outcome of
independent variable on the responses.
3. Results and discussion
3.1. Greywater characterization
Greywater from domestic households is very difficult to
treat successfully because of their high compositional
Figure 1. Reduction in Turbidity and Electrical conductivity (EC) after treatment with consortium.
variability [5]. Assembled consortium (M. luteus, R. equi
and A. niger) has been shown to possess tremendous
potential for pollutant degradation in greywater. Grey-
water without any prior treatment was obtained directly
8 from the households. Preliminary characterization was
35
200 done in terms of organic load. The pH was slightly
1:3 acidic i.e. 6.2 and turbidity reported was 40 NTU. The
colour and order was milky and pungent, respectively
as judged by the senses of sight and smell. The electrical
conductivity was 2048 μS/cm and total solid (TS) con-
centration was 1910 mg L−1. Initial COD, oil & grease
and sulphate values were 1165.6, 58 and 95.6 mg L−1,
respectively. Microbial growth and degradation exper-
(3) iments were carried out at shaking and static conditions,
respectively. The pH of the greywater shifts towards
neutral pH after treatment Table 1. The graphical rep-
resentation shows the reduction in turbidity 1.30 NTU.
It may be due to the excess consumption of suspended
solids, by micro-organisms, as their macro-nutrients for
further growth. Similar decrease in the turbidity of
wastewater was reported by Porwal et al. with the con-
sortium of mixed bacterial culture [36]. The electrical
conductivity was 480.70 μS/cm after treatment, as rep-
resented in Figure 1. The observed reduction in EC
might be due to the use of ions by micro-organisms
for their growth and survival. Figure 2 shows the
reduction in TDS and TSS. The organic contents along
with other mineral ions of greywater could have been
metabolized by the micro-organisms to cause overall
reduction of total solids which also signifies the
efficiency of the consortium in the bioremediation
process. Gaikwad et al. had reported similar reduction
in TSS and TDS by using microbial consortia of various
bacterial species, namely Pseudomonas, Actinomycetes,
Bacillus, Staphylococcus and Streptomyces [37]. Figure 3

Figure 2. Reduction in Total suspended solids (TSS) and Total dissolved solids (TDS) after treatment with consortium.
shows the performance of consortium in terms of
reduction in BOD and TOC of the greywater. The result
showed that the consortium accounted for the
maximum removal (454.5 mg L−1) of BOD and
(96.23 mg L−1) of TOC in 96 h. The consortium has
shown slight reduction in BOD and TOC during initial
hours because of its negligible growth during this
time. Considerable reduction in BOD and TOC has also
been reported by Porwal et al. and Gaikwad et al. from
wastewaters by using bacterial isolates [36,37]. It is
also evident from Figure 4 that after consortium treat-
ment, significant reduction in heavy metal concentration
(Fe and Mn) is being recorded and similar such obser-
vation was reported by Asare et al. [38]. However, con-
centration of heavy metals is in general low for
untreated greywater samples according to the drinking
Figure 3. Reduction in Biochemical oxygen demand (BOD) and Total Organic carbon (TOC) after treatment with consortium.
ENVIRONMENTAL TECHNOLOGY 5
water quality guidelines – Drinking Water Directive
2015/1787/EC and WHO (2008). The degradation
dynamics of the three significant parameters of grey-
water viz., COD, oil & grease and sulphate are further ela-
borated and discussed separately by applying statistics.
3.2. Process optimization for pollutant removal
from greywater stream
The experimental and predicted results of 30 runs, for a
study on the effects of four independent variables (pH,
Temperature (°C), Inoculum size (µL) and C/N ratio) on
the COD, oil & grease and sulphate removal, are pre-
sented in Table 3. The data for different coefficients
and ANOVA are presented in Table 4. The correlation
coefficients of R 2 were 0.9140–0.9499 for all the
6 N. RAJPAL ET AL.

Figure 4. Reduction in heavy metals, Iron (Fe) and Lead (Pb) after treatment with consortium.

responses, which shows that the equations were reliable.
According to Abbasi et al. [39], for a good fitness of
model, R 2 should be at least 0.8. Darvishmotevalli et al.
and Olmez reported that high R2 values suggest a
great accordance between the experimental data and
data estimated by the model [40,41]. Moreover, the F-
value of 9.91–17.59 and P-value of 0.0001 indicate that
the obtained model is significant, confirming the
qualification of the model to predict the COD, oil &
grease and sulphate removal from greywater by
microbial consortium. These values indicate that the
regression model layout gives a good explanation of
the association between the four factors and the
response. Ahmadi et al. and Muhamad et al. reported
similar such regression model along with their factors
and responses [42,43]. Taken into account the

Table 3. Box Behnken design-based experimental conditions for the treatment of greywater using bacterial–fungal consortium.
%COD %Oil & Grease %Sulphate
Run No. pH Temperature Inoculum size C/N ratio Experimental Predicted Experimental Predicted Experimental Predicted
1 −1 1 0 1 73.5 74.30 65.3 65.62 64.7 67.97
2 0 0 1 −1 64.8 63.55 58.4 58.80 64.3 69.18
3 −1 0 0 0 64.6 62.37 60.4 54.71 68.6 65.92
4 −1 0 −1 1 65.3 63.28 58.4 61.45 65.4 66.03
5 0 −1 −1 1 59.2 57.19 49.4 47.76 57.9 58.84
6 0 0 −1 0 63.2 66.91 57.9 58.71 65.7 65.92
7 1 0 −1 1 64.3 65.74 55.6 57.37 64.2 62.08
8 0 −1 1 1 59.2 57.92 49.4 45.69 58.9 55.45
9 −1 0 1 1 65.3 64.15 58.4 60.94 65.4 65.66
10 0 1 0 −1 68.5 68.78 62.7 64.83 69.6 67.63
11 0 0 0 1 62.5 62.65 59.7 59.61 64.9 66.10
12 0 0 0 1 62.5 62.65 59.7 59.61 64.9 66.10
13 1 0 1 1 62.5 64.82 64.5 65.76 75.8 73.31
14 1 1 0 1 76.4 76.32 72.4 72.33 76.4 80.73
15 0 1 −1 1 78.4 76.92 76.6 74.78 68.7 68.90
16 0 1 1 1 78.7 77.95 82.6 78.71 89.7 85.51
17 0 0 0 1 62.5 62.65 59.7 59.61 64.9 66.10
18 −1 −1 0 1 49.6 52.14 49.7 50.99 54.5 55.28
19 0 1 0 0 78.4 75.92 65.5 68.83 72.5 70.85
20 1 0 0 −1 59.3 58.77 57.1 57.26 63.8 63.23
21 1 −1 0 1 57.6 55.56 45.2 46.09 56.7 58.53
22 0 0 −1 −1 56.3 56.66 65.1 62.93 61.8 61.92
23 0 0 0 1 65.1 64.95 60.6 60.69 79.6 78.40
24 −1 0 0 −1 55.2 53.56 62.5 60.99 59.7 57.44
25 0 −1 0 −1 49.5 52.27 44.1 45.08 57.6 57.39
26 0 0 0 1 65.1 64.95 60.6 60.69 79.6 78.40
27 0 −1 0 0 51.8 51.81 46.6 48.78 58.4 58.51
28 1 0 0 0 61.4 60.28 63.2 59.18 64.8 63.82
29 0 0 1 0 59.7 61.80 61.3 64.69 66.9 71.88
30 0 0 0 1 65.1 64.95 60.6 60.69 79.6 78.40
 ENVIRONMENTAL TECHNOLOGY 7

Table 4. ANOVA for the percent reduction of pollutants in greywater.

Responses Source Sum of squares df Mean square F-Value P-value Prob > F
% COD reduction Model 1297.73 14 92.7 17.59 <0.0001 Significant
Residual 68.51 13 5.27
Lack of fit 68.51 9 7.61
Pure error 0.000 4 0.000
r2 0.9499
Adjusted r 2 0.8959
CV% 3.61
% Oil & grease reduction Model 1571.60 14 112.26 9.91 <0.0001 Significant
Residual 147.23 13 11.33
Lack of fit 68.51 9 7.61
Pure error 0.000 4 0.000
r2 0.9143
Adjusted r 2 0.8221
CV% 5.63
% Sulphate reduction Model 1621.38 14 115.81 9.87 <0.0001 Significant
Residual 152.47 13 11.73
Lack of fit 152.47 9 16.94
Pure error 0.000 4 0.000
r2 0.9140
Adjusted r 2 0.8215
CV% 5.12
DF: degrees of freedom of variance source; F: F-value of variance source; P: probability of error to be significant.

dependability of quadratic terms on linear terms, with all
the linear coefficients regardless of their significance
included, the following second-degree polynomial
equation for COD, oil & grease and sulphate reduction
can be obtained and given as follows:
% COD = +62.00 + 2.23∗ A + 9.88∗ B + 1.96∗ C
+ 3.26∗ D − 0.16∗ AB + 0.10∗ AC
− 1.19∗ AD + 0.000∗ BC + 1.26∗ BD
− 1.51∗ CD − 1.06∗ A2 + 2.44∗ B2
+ 1.37∗ C2 − 1.09∗ D2
% Oil &Grease = +57.41 − 1.32∗ A + 12.71∗ B
+ 0.98∗ C + 0.90∗ D + 1.66∗ AB
+ 0.72∗ AC + 3.571E − 003∗ AD
+ 1.50∗ BC − 0.72∗ BD
− 0.51∗ CD + 0.77∗ A2
− 0.39 B2 + 5.52∗ C2
− 0.34∗ D2
Adjusted R-squared value should be in close proxi-
mity to R-squared since it rectifies R-squared value for
the sample size and the number of terms in the
model. Predicted R-squared must be in the range of
0.2 from adjusted R-squared. In the present study,
adjusted R-squared and predicted R-squared are in the
reasonable values indicating the adequacy of the
model. In similar kinds of optimization studies, Nair
et al.[44] and Ghafoori et al. [45] reported that predicted
R-squared must be 0.2 from adjusted R-squared denoted
that the regression models were well fitted. For reprodu-
(4) cibility of model the value of the coefficient of variation
(CV) should be less than 10. The value for CV is 3.61–5.12
which is suggestive of a good model fit since the lower
the CV, the smaller the residuals relative to the predicted
value. These findings are consistent with data reported
in previous studies [42,46–48]. To consider the relation-
ship between the predicted values from the model
values calculated by Equations (4)–(6) and the observed
values, the data obtained were very close to linear, indi-
cating that both values were accurate and reliable, as
(5) shown in Figure 5.

% Sulphate = +68.23 − 5.28∗ A + 10.65∗ B

+ +5.95∗ C + 4.67∗ D + +6.04∗ AB
+ 3.49∗ AC + −3.77∗ AD
+ 5.00∗ BC + 0.56∗ BD − 2.65∗ CD
− 2.57∗ A2 − 1.57B2 + 1.47∗ C2
− 1.92∗ D2
where A, B, C and D were the coded values of the
process variables, pH, temperature (°C), inoculum size
(µL) and C/N ratio, respectively.
3.3. Interactive effects of factors on responses
Response surface as a function of factors can be plotted
in order to exhibit the outcome of factors on the depen-
dent variable. The three-dimensional response plots are
(6) the graphical representations of the regression
equation. The relationship between % COD, oil &
grease and sulphate and the four independent factors
(pH, temperature, inoculum size and C/N ratio) is
shown through 3D surface plots (Figures 6–8). The
8 N. RAJPAL ET AL.

Figure 5. Regression plots of the Predicted values against the Actual values from the response surface models describing (a) COD
reduction (b) Oil & Grease reduction and (c) Sulphate reduction using microbial consortium.

effect of each variable was further estimated by the use
of three-dimensional response plots to show how the
response changes as each variable moves from the
chosen reference point, with all other factors held at
constant reference values [49].
Figure 6(a) shows the interactive effect of two vari-
ables pH (A) and temperature (B) on percent removal
of COD. COD reduction increases with the increase in
pH and then decreases with further increase of pH. In
the case of temperature, less reduction can be seen at
 ENVIRONMENTAL TECHNOLOGY 9

Figure 6. 3D plot showing the interactive effect of (a) pH and temperature(b) pH and inoculum size(c) inoculum size and temp(d) pH
and C/N ratio(e) C/N and temperature(f) inoculum size and C/N ratio for COD degradation by using microbial consortium.

lower temperature and increases with the increase in Figure 6(b) the degradation of COD increases with the
temperature. Maximum COD degradation was found to increase in inoculum size and maximum results were
be 77.9% at pH 7 and temperature 35°C (Table 4). In obtained at 150 µL. Similar patterns of degradation

Figure 6 Continued
10 N. RAJPAL ET AL.

Figure 7. 3D plots showing the interactive effect of (a) pH and temperature (b) pH and inoculum size (c) inoculum size and temp (d)
pH and C/N ratio (e) C/N and temperature (f) inoculum size and C/N ratio for oil & grease degradation by using microbial consortium.

activity can be seen in (Figure 6(c–f)). From the figures, it
is inferred that percentage removal of COD increases
with the increase in pH, temperature, inoculum size
and C/N ratio. Similar patterns were obtained for the
effect of process variables on oil & grease and sulphate
reduction in greywater and are depicted in (Figures 7
and 8). The maximum predicted values for degradation
of COD, oil & grease and sulphate were found to be
77.9, 78.7 and 85.5%, respectively under optimum
conditions.
3.4. Response prediction, model validation and
confirmation
For validation of obtained combination of variables
through RSM, It is necessary to check the adequacy of

Figure 7 Continued
 ENVIRONMENTAL TECHNOLOGY 11

Figure 8. 3D plot showing the interactive effect of (a) pH and temperature (b) pH and inoculum size (c) inoculum size and temp (d) pH
and C/N ratio (e) C/N and temperature (f) inoculum size and C/N ratio for sulphate degradation by using microbial consortium.

the obtained model before analysing and predicting the
responses. The obtained model should show approxi-
mation to the actual test results, otherwise it may lead
to the poor results. The confirmatory experiments were
applied on the predicted optimized conditions (Table
3) to establish the validity and acceptability of the
models. Out of the all conditions applied, one solution
was then chosen for further process studies to confirm
the validity of the statistical experimental strategies
with experimental data. The optimal reduction con-
ditions were as follows: pH 7, temperature, 35°C, inocu-
lum size, 150 µL and C/N ratio, 1:2. The average

Figure 8 Continued
12 N. RAJPAL ET AL.
reduction in the pollutants (COD, oil & grease and sul-
phate) was 78.7%, 82.6% and 89.7% after 96 h of incu-
bation. The observed and predicted results found were
in close agreement with each other and hence validated
the findings of response surface optimization lead to the
acceptability and reliability of the obtained model. The
obtained results are in line with the earlier reports
studied by various researchers regarding reliability of
the obtained model [42,46,47].
4. Conclusions
Microbial consortia are known to be a better efficient
means to treat contaminated waste waters. Although
the individual microbial species in the consortia
possess varied food demands, substrate consumption
capacity and pollutant affinity properties, their combi-
nation is certainly proved to exhibit a much efficient
coordination and synergy in terms of mediating the
crucial chemical degradative biochemical pathways. In
the present study, the degradation efficiency of a
novel microbial consortium, developed for the reduction
of various pollutants from greywater, is being evidently
observed. BBD was employed to study and optimize
the process variables under different operating con-
ditions, such as pH; temperature; inoculum size and C/
N ratio to treat greywater by using novel microbial con-
sortium. From the results, it’s clear that the operating
variables have a significant effect on the pollutant
reduction efficacy. The present study marks the potential
of novel consortium for the bioremediation of greywater
under optimized conditions, indicating the ideal con-
ditions for degradation of pollutants present in grey-
water. The obtained results are indicative of the
potential applications of the novel microbial consortium,
which can be further scaled up.
Acknowledgements
The authors are grateful to the Shriram Institute for Industrial
Research, Bangalore and Karnataka, India for carrying out
experimental work in their research laboratories.
Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Ananda Vardhan Hebbani http://orcid.org/0000-0003-1977-
6098
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