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RESEARCH ARTICLE
Exploring the efficacy of wastewater-grown microalgal biomass
as a biofertilizer for wheat
Nirmal Renuka
1
& Radha Prasanna
2
& Anjuli Sood
2
& Amrik S. Ahluwalia
1
&
Radhika Bansal
3
& Santosh Babu
2
& Rajendra Singh
4
& Yashbir S. Shivay
3
& Lata Nain
2
Received: 10 May 2015 /Accepted: 25 November 2015
#
Springer-Verlag Berlin Heidelberg 2015
Abstract Microalgae possess the ability to grow and glean
nutrients from wastewater; such wastewater-grown biomass
can be used as a biofertilizer for crops. The present investiga-
tion was undertaken to evaluate two formulations (formula-
tion with unicellular microalgae (MC1) and formulation with
filamentous microalgae (MC2); T4 and T5, respectively), pre-
pared using wastewater-grown microalgal biomass, as a
biofertilizer (after mixing with vermiculite/compost as a car-
rier) in wheat crop (Triticum aestivum L. HD2967) under con-
trolled conditions. The highest values of available nitrogen
(N), phosphorus (P), and potassium (K) in soil and nitrogen-
fixing potential were recorded in treatment T5 (75 % N+full-
dose PK+formulation with filamentous microalgae (MC2).
Microbial biomass carbon was significantly enhanced by
31.8–67.0 % in both the inoculated treatments over control
(recommended dose of fertilizers), with highest values in T4
(75 % N+full-dose PK+formulation with unicellular
microalgae (MC1)). Both the microalgal formulations signif-
icantly increased the N, P, and K content of roots, shoots, and
grains, and the highest total N content of 3.56 % in grains was
observed in treatment T5. At harvest stage, the treatments
inoculated with microalgal formulations (T4 and T5) recorded
a7.4–33 % increase in plant dry weight and up to 10 % in
spike weight. The values of 1000-grain weight showed an
enhancement of 5.6–8.4 %, compared with T1 (recommended
doses of fertilizers). A positive correlation was observed be-
tween soil nutrient availability at mid crop stage and plant
biometrical parameters at harvest stage. This study revealed
the promise of such microalgal consortia as a biofertilizer for
25 % N savings and improved yields of wheat crop.
Keywords Microalgae
.
Microbial biomass carbon
.
Nutrient
availability
.
Soil fertility
.
Plant nutrition
.
Yield
Introduction
Wastewaters represent rich sources of nitrogen and phospho-
rus, with promise for use as low-cost media for microalgae.
These organisms possess the ability to flourish in diverse
types of environments and assimilate such nutrients. Such a
method of biomass cultivation is beneficial, as assimilation of
nitrogen and phosphorus from such wastewaters can help in
recycling this algal biomass as a biofertilizer and, at the same
time, reduces the use of chemical fertilizers and sewage dis-
posal (Pittman et al. 2011). India is one of the largest pro-
ducers and consumers of fertilizers in the world, but the grad-
ual increase in the fertilizer consumption pattern in the country
has raised not only serious environmental concerns but also is
insufficient with the current industrial capacity. It has been
estimated that by 2020, the fertilizer demand would increase
to about 41.6 million tons, inclusive of 23.0, 11.5, and
7.1 million tons of nitrogen (N), phosphorus (P), and potassi-
um (K), respectively (Jaga and Patel 2012). A major drawback
Responsible editor: Zhihong Xu
Electronic supplementary material The online version of this article
(doi:10.1007/s11356-015-5884-6) contains supplementary material,
which is available to authorized users.
* Radha Prasanna
radhapr@gmail.com
1
Department of Botany, Panjab University, Chandigarh 160014, India
2
Division of Microbiology, ICAR-Indian Agricultural Research
Institute, New Delhi 110012, India
3
Division of Agronomy, ICAR-Indian Agricultural Research Institute,
New Delhi 110012, India
4
Water Technology Centre, ICAR-Indian Agricultural Research
Institute, New Delhi 110012, India
Environ Sci Pollut Res
DOI 10.1007/s11356-015-5884-6
regarding the use of chemical fertilizers is the environmental
pollution, due to the presence of heavy metals (Hg, Cd, As,
Pb, etc.) and natural radionuclides (U
238
,Th
232
, etc.) (Savci
2012).
The availability of macro- and micronutrients is an impor-
tant factor in achieving higher crop yields. N, P, and K are the
primary essential macronutrients, which play a crucial role in
crop nutrition. Microalgal biomass contains a higher amount
of nitrogen, and cyanobacterial members also have an ability
to fix atmospheric nitrogen; therefore, such microalgae/
cyanobacteria can serve as biofertilizers in various cropping
systems. Biofertilizers include micro/macroorganisms which
can colonize the soil, rhizosphere, or the interior of the plant
and improve the growth and nutrition of the plant (Bashan
1998; Prasanna et al. 2012a, b). Different types of microbial
biofertilizers are available such as carrier based or liquid
formulations or pellets (Motsara et al. 1995;Lataetal.2002;
Yan et a l. 2013).
The role of algae in agricultural systems as biofertilizers is
well known (Venkataraman 1972;Kaushik1998;
Vaishampayan et al. 2001; Prasanna et al. 2012a, b).
However, they also have an important role in reducing erosion
of soil by regulating the water flow into soils and improving
soil fertility, besides playing a role in the reclamation of waste-
lands, saline soils, etc. (Prasanna et al. 2008; Zhan and Sun
2012). and biocontrol of agricultural pests (Chaudhary et al.
2012; Prasanna et al. 2014). They also play a key role in the
formation of microbiotic crusts (Mazor et al. 1996; Colica
et al. 2014) as well as wastewater treatment (de-Bashan et al.
2004; Renuka et al. 2015). The use of microalgae, especially
cyanobacteria, as a biofertilizer is not new (Singh et al. 2011).
and scattered reports on the use of green algae as a biofertilizer
are also available (de-Bashan and Bashan 2004). Members of
cyanobacterial genera Nostoc, Anabaena,and
Cylindrospermum are found to form loose or tight associations
with various members of the Plant Kingdom, including crop
plants (Rai et al. 2000). The colonization of wheat by
nitrogen-fixing cyanobacteria and their ability to form useful
associations are well investigated (Gantar et al. 1991;
Karthikeyan et al. 2009;Soodetal.2011;Babuetal.2015).
Most of reports available on microalgal biofertilizers are based
on cyanobacteria (Venkataraman 1972; Karthikeyan et al.
2007; Manjunath et al. 2011; Prasanna et al. 2013, 2014).
Microalgae increase the soil nutrient availability to the plants
and act as plant growth promoters, due to the release of growth
hormones (Manjunath et al.
2011;R
anaetal.2012). Certain
cyanobacteria also improve the growth by increasing the en-
dogenous hormones of the plant (Hussain and Husnain 2011).
Microalgal biomass contains a high amount of proteins
and, for their proliferation, requires high amounts of nitrogen
(8–16 tons N ha
−1
) as a fertilizer (Markou and Georgakakis
2011). However, microalgae can grow in different types of
wastewater, playing dual roles of nutrie nt/contaminant
sequestration and production of useful biomass (Bhatnagar
et al. 2010; Renuka et al. 2013a, b). However, the utilization
of wastewater-grown biomass is questionable due to the accu-
mulation of toxic heavy metals, if present above safe limits,
although trace amounts can serve as micronutrients. A major
drawback of wastewater is the presence of pathogenic bacte-
ria, but microalgal growth is known to increase the pH of the
media and suppress the growth of bacteria (Toha et al. 1991).
This illustrates that sewage-grown microalgae can have an
edge for the direct use of sewage sludge or wastewater in
agronomic practices.
Wheat is the leading source of protein for human diet,
because of its higher protein content than other cereals (rice
and maize), but consumes a major share of chemical fertil-
izers. The deployment of microbial biofertilizers in cereal
crops is beneficial, as they not only reduce the use of chemical
fertilizers but also improve the overall health and nutritional
status of soil (Singh et al. 2011;Prasannaetal.2013).
However, no published reports exist on using wastewater-
grown microalgae as biofertilizers (Cabanelas et al. 2013).
Therefore, the hypothesis of th e present study was—can
sewage-grown microalgal biomass be useful as a biofertilizer
for wheat crop.
Materials and methods
Details of microalgal consortia and growth
Two microalgal consortia, viz. MC1 comprising native unicel-
lular strains of sewage (species of Chlorella, Scenedesmus,
Chlorococcum, Chroococcus) and MC2 that is made up of
native filamentous strains isolated from sewage wastewater
(species of Phormidium, Anabaena, Westiellopsis,
Fischerella, Spirogyra), were evaluated for their potential to
be used as a biofertilizer. The isolation, screening, and selec-
tion towards the development of consortia have been de-
scribed in our earlier studies, and these microalgal consortia
had shown promise in wastewater remediation (Renuka et al.
2013a, b, 2014). Based on earlier studies, the microalgal con-
sortia were grown in full-strength wastewater and harvested
on the 6th day of growth (Renuka et al. 2013b). The harvested
microalgal biomass was dried at 60 °C and was used in the
preparation of formulations.
Experimental setup of wheat crop
The experiment was carried out in pots of “8 in. size” contain-
ing 6 kg sterile soil, planted with wheat (Triticum aestivum L.)
variety HD2967. A total of five treatments were taken, viz. T1
(recommended/full dose of fertilizer of nitrogen (N)/phospho-
rus (P)/potassium (K) in 120:60:60 kg ha
−1
), T2 (75 % N+
full-dose PK), T3 (75 % N+full-dose PK+carrier), T4 (75 %
Environ Sci Pollut Res
N+full-dose PK+MC1), and T5 (75 % N+full-dose PK+
MC2). Initial physicochemical properties of soil are tabulated
in Supplementary Table 1. Pots were kept in the controlled
environment chamber of the National Phytotron Facility,
ICAR-IARI, New Delhi, which provided optimal temperature
conditions (24±2 and 20±2 °C; day and night temperature,
respectively) for the growth of wheat. The formulations were
prepared by mixing dry biomass of mic roalgal consortia
(20 μg c hlorophyll g
−1
carrier) using vermiculite/compost
(1:1) as a carrier. Water was added to maintain 60 % water
holding capacity of vermiculite/compost (1:1), and formula-
tions were stored at room temperature. Fifty grams of each
carrier-based formulation of MC1 and MC2 was mixed with
6 kg soil in T4 and T5, respectively, while in T3, 50 g of carrier
was mixed with 6 kg of soil. Physicochemical characteristics of
the carrier used are given in our previous study (Chaudhary
et al. 2012). while characteristics of microalgal formul ations
(with carrier) are given in Supplementary Table 2. Pots contain-
ing soil were irrigated regularly to maintain the 60 % water
holding capacity. Before sowing, seeds were soaked in distilled
water for 24 h under dark conditions. Five seeds of wheat were
sown in each pot with equal spacing, and there were six pots for
each treatment. At the time of sowing, 50 % N was sup ple-
mented in T1 control and other treatments, respectively, wh ile
the remaining dose was supplied at the tillering stage.
Plant and soil samples were collected at mid (60 days after
sowing) and harvest stage by harvesting three replicate pots at
each stage. A minimum of three plants from each pot were
taken for evaluation of plant-related parameters. Soil cores
from the root region (0–15 cm diameter) were collected in
triplicate using auger from each pot for the assessment of
microbiological parameters.
Analyses of soil-related parameters
Microbial biomass carbon (MBC) in soil samples was mea-
sured using the extraction and estimation procedure described
in Voroney et al. (1993) and Jenkinson and Powlson (1976)
and expressed in microgram C per gram soil. Estimation of
ethylene as an index of nitrogenase activity was done using a
gas chromatograph (Bruker 450-GC) by measuring acetylene-
reducing activity (ARA). Ethylene (1000 ppm; Sigma Gases
and Services, New Delhi, India) was used for quantification in
samples t aken from vials containing soil cores (0–15 cm
depth), while similar vials with equivalent water were served
as control. The column temperature was maintained at 70 °C,
while injector and detector temperature was maintained at 150
and 250 °C, respectively. ARA values were expressed in
nanomoles of ethylene produced per gram soil per hour.
Available nitrogen (N) in soil samples was estimated using
the method of Subbiah and Asija (1956). while estimation of
available phosphorus (P) was carried out using the method of
Olsen et al. (1954). For the estimation of potassium (K),
extraction was carried out in ammonium acetate and concen-
tration was measured using a flame photometer (Prasad et al.
2006).
Analyses of plant-related parameters
For the analyses of macronutrients (N, P, and K) in wheat,
plant samples (grain, shoot, and root, separately) were ground
and digested. Total nitrogen in the plant samples was mea-
sured using the Kjeldahl method described by Prasad et al.
(2006). Phosphorus concentration in plant samples was esti-
mated using the method of Jackson (1958). Potassium con-
centration was measured with a flame photometer as described
in Prasad et al. (2006).
Plant biometrical parameters, viz. fresh and dry weight,
shoot and root length, and number of tillers, were calculated
at mid crop stage, while plant height, plant dry weight, straw
yield, spike weight, and weight of grains were measured at the
time of crop harvest.
Statistical analyses
The results were statistically analyzed for their significance
using the Statistical Package for the Social Sciences (SPSS
version 16.0). The differences among various treatments were
evaluated using one-way analysis of variance (ANOVA). The
triplicate sets of data were analyzed in accordance with the
experimental design (completely randomized design). Post
hoc least significant differences (LSDs) calculated at a P level
of 0.05 was used to evaluate the comparisons between the
different means and represented as critical difference (CD)
values in the tables. Duncan’s multiple range test (DMRT)
analysis was rep resented as superscripts in tables, with a
representing highest values. Similar superscripts indicate
non-significantly (p≤ 0.05) different mean values. Standard
deviation (SD) values are depicted in the graphs as error bars.
Interrelationships between various soil- and plant-related pa-
rameters were evaluated using correlation coefficients calcu-
lated with Microsoft Excel package. Correlation coefficients
were analyzed for their significance by means of Pearson’s
tables.
Results
Soil nutrient analyses at mid crop and harvest stages
Analyses of soil samples at mid crop stage revealed higher
available N, P, and K content in microalgae-inoculated treat-
ments in comparison with the other treatments ( Table 1).
Signi ficantly higher values of available N (199.37 kg ha
−1
)
were observed in T5 (75 % N+full-dose PK+MC2).
However, the values of available N content in treatments T4
Environ Sci Pollut Res
(75 % N+full-dose PK+MC1), T5 (75 % N+full-dose PK+
MC2) and T1 (recommended dose of NPK) were not signifi-
cantly different. Lowest N content of 141.15 kg ha
−1
was
observed in T2 (75 % N+full-dose PK). T5 revealed a
significantly higher P content of 28.37 kg ha
−1
compared
with other treatments; however, the values were not sig-
nificantly different as compared with T4. A significantly
higher amount of available K (521.17 kg ha
−1
)was
found in T5 (75 % N+full-dose PK+MC2). The avail-
able K content from T4 and T1 control s amples did not
exhibit significant differences. The lowest value of avail-
ableK(440.2kgha
−1
) a t mid crop stage was seen in T3
(75 % N+full-dose PK+carrier).
Soil characteristics at harvest stage revealed a de-
crease in the a vailability of nutrient s in all the treat-
ments, compared with mid crop stage (Table 1). The
highest amount of N at harvest was observed in T5
(75 % N+full-dose PK+MC2). Availab l e P conte nt at
harvest stage revealed higher values (21.65 kg ha
−1
)in
T5, which was not significantly higher than T4 (75 %
N+full-dose PK+MC1). T2 (75 % N+full-dose PK)
samples exhibited lowest available P of 3.73 kg ha
−1
.
Samples from T5 and T4 revealed significantly higher
available K of 455.09 and 439.04 kg ha
−1
, respectively,
in soil, compared with other treat ments.
Soil biologi cal parameters at mi d crop stage
The ARA values (index of nitrogen fixation) revealed a
wide variation among the different treatments. T5 (75 %
N+full-dose PK+MC2) recorded significantly higher
ARA values of 5.86 nmol g
−1
ha
−1
, while T1 (recom-
mended dose of NPK) showed the lowest values
(Fig. 1a). Microalgal consortium-inoculated treatments,
viz. T5 and T4, recorded 31.8 and 67.0 % higher MBC
as compared with T1 (recommended dose of NPK), re-
spectively, with highest values of 353.82 μgCg
−1
soil ha
−1
in T4 (Fig. 1b).
Plant nutrient characteristics at mid crop and harvest
stages
Higher nitrogen content of 2.18 and 4.51 % in roots and
shoots was recorded in T5 (75 % N+full-dose PK+MC2) at
mid crop stage, in comparison with other treatments (Table 2).
T2 (75 % N+full-dose PK) recorded lowest N content
(1.14 %) in roots, while in shoots, the lowest N content
(3.03 %) was observed in T3 (75 % N+full-dose PK+carrier).
At harvest stage, higher accumulation of N, i.e., 1.66 and
1.40 % in roots and shoots, respectively, was observed in T4
(75 % N+full-dose PK+MC1). However, N content in shoots
in T5, T4, and T1 did not vary significantly. Lowest total N in
roots and shoots was observed in T2 (75 % N+full-dose PK).
Total N content in grains revealed significantly higher values
for total N (3.56 %) in T5, compared with other treatments.
During the mid crop stage, highest total P content of
0.39 % in roots was recorded in T5 (75 % N+full-dose
PK+MC2); however, no significant differences in total P
content in shoot were recorded (Table 3). Total P content
of 0.61 % in grains was recorded in T4 (75 % N+full-
dose PK+MC1), which was significantly higher than T1
control (recommended dose NPK) and T5 (75 % N+full-
dose PK+MC2).
The K content in roots at mid crop stage revealed
higher values in T4 and T5—microalgal conso rtium-
inoculated treatments (Table 4). Highest K (1.43 %) in
roots was recorded in T5 (75 % N+full-dose PK+MC2)
followed by T4 (75 % N+full-dose PK+MC1), which
were significantly higher than the other treatments.
Higher accumulation of K, equivalent to 3.68 and
Table 1 Comparative account of nitrogen (N), phosphorus (P), and potassium (K) in soil in different treatments at mid crop and harvest stage of wheat
crop
Available N (kg ha
−1
) Available P (kg ha
−1
) Available K (kg ha
−1
)
Mid crop stage Harvest stage Mid crop stage Harvest stage Mid crop stage Harvest stage
T1 (recommended dose of NPK) 183.70±1.30
a
141.2±12.10
b
14.56±1.29
b
9.71±1.29
b
463.68±36.96
b
369.6±20.74
b
T2 (75 % N+full-dose PK) 141.15±12.55
c
106.65±3.15
c
8.21±1.29
c
3.73±1.29
c
453.60±29.63
c
365.12±39.60
b
T3 (75 % N+full-dose PK+carrier) 169.35±3.15
b
112.9±15.70
c
16.80±1.12
b
10.45±1.29
b
440.16±12.11
c
377.81±32.41
b
T4 (75 % N+full-dose PK+MC1) 186.30±10.1
a
146.93±7.00
b
26. 88±1.58
a
18.67±1.29
a
476.00±10.68
b
439.04±5.82
a
T5 (75 % N+full-dose PK+MC2) 199.37±10.36
a
162.30±2.50
a
28.37±1.29
a
21.65±2.59
a
521.17±25.25
a
455.09±12.34
a
CD (p=0.05) 15.24 15.27 2.04 2.82 39.77 34.86
Values are given as mean±standard deviation; Superscripts in lower case (a,b, etc.) represent DMRT (Duncan's Multiple Range Test) ranking along the
columns; MC1=species of Chlorella, Scenedesmus, Chlorococcum, and Chroococcus; MC2=species of Phormidium, Anabaena, Westiellopsis,
Fischerella,andSpirogyra
CD critical difference
Environ Sci Pollut Res
3.35 % in roots at ha rv es t s ta ge , w a s r eco r ded in T 4 an d
T5, respectively. Similarly, higher K content in shoots
was also recorded in microalgal consortium-inoculated
treatments. The K content of grains revealed significantly
higher values in T4 and T 5 (Table 4). Lowest K content
of 0.15 % in grains was recorded in T2 (75 % N+full-
dose PK).
Plant biometrical parameters
At mid crop stage, significantly higher fresh and dry weight of
16.75 and 3.75 g was observed in T4 (75 % N+full-dose PK+
MC1), respectively (Fig. 2a). The lowest fresh weight of 7.91 g
was rec orded in T2 (75 % N+full-dose PK) and dry weight of
2.31 g in T3 (75 % N+full-dose PK+carrier), respectively.
0.0
2.0
4.0
6.0
8.0
Full dose NPK 75%N + Full
dose PK
75%N + Full
dose PK +
carrier
75%N + Full
dose PK +
MC1
75%N + Full
dose PK +
MC2
ARA
(nmol ethylene g
-1
soil h
-1
)
Treatment
0
70
140
210
280
350
420
Full dose NPK 75%N + Full
dose PK
75%N + Full
dose PK +
carrier
75%N + Full
dose PK +
MC1
75%N + Full
dose PK +
MC2
MBC
(µg C g
-1
soil h
-1
)
Treatment
a
b
Fig. 1 Analyses of various soil
microbiological parameters, as
influenced by microalgal
consortia, at mid crop stage. a
Acetylene-reducing activity
(ARA). b Microbial biomass
carbon (MBC)
Table 2 Comparative account of nitrogen (N) in plant in different treatments at mid crop and harvest stages of wheat crop
Treatment Total N (%)
Mid crop stage Harvest stage
Root Shoot Root Shoot Grain
T1 (recommended dose of NPK) 1.30±0.18
b
3.28±0.23
b
1.56±0.04
a
1.38±0.06
ab
2.74±0.08
c
T2 (75 % N+full-dose PK) 1.14±0.29
b
3.11±0.31
b
0.67±0.08
c
1.21±0.10
c
2.63±0.03
c
T3 (75 % N+full-dose PK+carrier) 1.40±0.30
b
3.03±0.16
b
0.73±0.04
c
1.33±0.05
ab
2.73±0.04
c
T4 (75 % N+full-dose PK+MC1) 1.99±0.19
a
4.06±0.31
a
1.66±0.06
a
1.40±0.08
a
3.22±0.06
b
T5 (75 % N+full-dose PK+MC2) 2.18±0.17
a
4.51±0.19
a
1.04±0.12
b
1.39±0.06
a
3.56±0.13
a
CD (p=0.05) 0.34 0.44 0.13 0.12 0.13
Values are given as mean±standard deviation; Superscripts in lower case (a,b, etc.) represent DMRT (Duncan's Multiple Range Test) ranking along the
columns; MC1=species of Chlorella, Scenedesmus, Chlorococcum, and Chroococcus; MC2=species of Phormidium, Anabaena, Westiellopsis,
Fischerella,andSpirogyra
CD critical difference
Environ Sci Pollut Res
Shoot length in all the treatments was not significantly different;
however , significantly higher root length of 8.33 and 8.83 cm
was recorded respectively in T4 and T5 treatments compared
with other treatments (Fig. 2b). Inoculation of formulated
microalgal consortia also enhanced th e number of tillers in wheat
plant with significantly higher values in T4 (75 % N+full-dose
PK+MC1).
Similartothemidcropstage,nosignificantdifferenceswere
recorded in plant height in different treatments at harvest (data
not shown). However , significantly higher plant dry weight of
7.20 g was recorded in T4 (75 % N+full-dose PK+MC1) at
harvest, compared with other treatments (T a ble 5). Inoculation
of formulations of microalgal consortia resulted in an increase
in the number of spikes and 1000-grain weight (Table 5,Fig.3).
The highest weight of spikes (1.43 g) and grain (38.67 g for
1000 grains) was recorded in T5 (75 % N+full-dose PK+
MC2),followedbyT4(75%N+full-dosePK+MC1).
Correlations among the soil/plant nutrient characteristics
and plant biometrical parameters
A positive correlation was recorded between the soil nutrient
characteristics at mid crop stage and the plant biometrical pa-
rameters at harvest stage. Plant dry weight at harvest stage was
positively correlated with available N (r=0.72) and P (r=0.81)
of soil at mid crop sta ge (Supplementary Fig. 1A, B). A strong
positive correlation was found between spike weight at harvest
stage and soil available N (r=0.99) and P (r=0.84) at mid crop
stage (Supplementary Fig. 1C, D). The 1000-grain weight
values correlated strongly with the soil available N (r=0.83),
Table 4 Comparative account of potassium (K) in plant in different treatments at mid crop and harvest stages of wheat crop
Treatment Total K (%)
Mid crop stage Harvest stage
Root Shoot Root Shoot Grain
T1 (recommended dose of NPK) 0.59±0.05
c
1.83±0.07
ab
1.90±0.10
d
1.17±0.01
bc
0.53±0.01
c
T2 (75 % N+full-dose PK) 0.48±0.11
c
1.79±0.34
c
1.92±0.12
d
1.03±0.05
d
0.14±0.02
c
T3 (75 % N+full-dose PK+carrier) 0.77±0.14
b
1.89±0.34
ab
2.81±0.16
c
1.11±0.03
c
0.33±0.02
d
T4 (75 % N+full-dose PK+MC1) 1.40±0.19
a
2.53±0.50
a
3.68±0.12
a
1.23±0.01
b
0.85±0.03
a
T5 (75 % N+full-dose PK+MC2) 1.43±0.14
a
2.48±0.37
ab
3.35±0.20
b
1.34±0.05
a
0.79±0.02
b
CD (p=0.05) 0.14 0.63 0.25 0.06 0.03
Values are given as mean±standard deviation; Superscripts in lower case (a,b, etc.) represent DMRT (Duncan's Multiple Range Test) ranking within the
column; MC1=species of Chlorella, Scenedesmus, Chlorococcum, and Chroococcus; MC2=species of Phormidium, Anaba ena, Westiellopsis,
Fischerella,andSpirogyra
CD critical difference
Table 3 Comparative account of phosphorus (P) in plant in different treatments at mid crop and harvest stages of wheat crop
Treatment Total P (%)
Mid crop stage Harvest stage
Root Shoot Root Shoot Grain
T1 (recommended dose of NPK) 0.14±0.004
b
0.52±0.04
ab
0.06±0.01
b
0.23±0.03
a
0.53±0.01
bc
T2 (75 % N+full-dose PK) 0.16±0.02
b
0.43±0.03
b
0.06±0.02
b
0.11±0.02
c
0.47±0.04
d
T3 (75 % N+full-dose PK+carrier) 0.17±0.03
b
0.43±0.01
b
0.10±0.02
a
0.15±0.03
bc
0.52±0.01
cd
T4 (75 % N+full-dose PK+MC1) 0.30±0.06
a
0.58±0.08
b
0.10±0.01
a
0.21±0.03
a
0.61±0.03
a
T5 (75 % N+full-dose PK+MC2) 0.39±0.10
a
0.54±0.05
b
0.08±0.01
ab
0.20±0.02
ab
0.56±0.01
b
CD (p=0.05) 0.09 0.08 0.025 0.05 0.04
Values are given as mean±standard deviation; Superscripts in lower case (a,b, etc.) represent DMRT (Duncan's Multiple Range Test) ranking within the
column; MC1=species of Chlorella, Scenedesmus, Chlorococcum, and Chroococcus; MC2=species of Phormidium, Anaba ena, Westiellopsis,
Fischerella,andSpirogyra
CD critical difference
Environ Sci Pollut Res
P(r =0.80), and K (r =0.90) at the mid crop stage
(Supplementary Fig. 1E, F).
A positive correlation was also found between plant
nutrient characteristics and biometrical parameters at the
harvest stage, especially straw yield with shoot total N
(r =0.87) and P (r = 0.93) content (Supplementary
Fig. 2A, B). Spike weight was also positively correlated
with shoot total N (r=0.97) and P (r=0.86) content at
harvest sta ge (Supplementary Fig. 2C, D). A positive
correlation was f ound between 1000-grain weight and
shoot total N (r =0.75), P (r=0.75), and K (r =0.92)
content (Supplementary Figs. 2E, F and 3A). Plant dry
weight and straw yield at harvest stage were positively
correlated with total N content of roots. A strong posi-
tive correlation was obtained for straw yield (r=0.95)
and plant dry weight (r=0.84) with root total N content
(Supplementary Fig. 3B, C). The nutrient content (N, P,
and K) of grains were directly related with soi l nutrient
content at mid crop stage. A positive correlation was
observed be tween g rain N (r=0.79), P (r =0.90), and
K(r=0.74) with available N, P, and K content of soil
at mid crop stage (Supplementary Fig. 3D,E,F).
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0.0
3.0
6.0
9.0
12.0
15.0
18.0
21.0
Full dose NPK 75%N + Full
dose PK
75%N + Full
dose PK +
carrier
75%N + Full
dose PK +
MC1
75%N + Full
dose PK +
MC2
Dry weight (g)
Fresh weight (g)
Treatment
Dry weight Fresh weight
0.0
2.0
4.0
6.0
8.0
10.0
Full dose NPK 75%N + Full
dose PK
75%N + Full
dose PK +
carrier
75%N + Full
dose PK +
MC1
75%N + Full
dose PK +
MC2
Root length (cm)
Treatment
a
b
Fig. 2 Comparative analyses of
the effect of application of
microalgal consortia on plant
biometrical parameters at mid
crop stage. a Fresh and dry
weight. b Root length
Table 5 Comparative account of various plant parameters in different treatments at mid and harvest stage of wheat crop
Treatment Number of tillers (mid crop stage) Plant dry weight (g) (harvest stage) Spike weight (g) (harvest stage)
T1 (recommended dose of NPK) 2.33±0.57
b
5.40±0.82
bc
1.30±0.08
ab
T2 (75 % N+full-dose PK) 1.33±0.57
c
4.22±0.23
c
0.88±0.05
c
T3 (75 % N+full-dose PK+carrier) 1.33±0.57
c
4.53±1.07
c
1.21±0.10
b
T4 (75 % N+full-dose PK+MC1) 3.00±0.00
ab
7.20±1.19
a
1.31±0.09
ab
T5 (75 % N+full-dose PK+MC2) 3.33±0.57
a
5.80±1.6
ab
1.43±0.15
a
CD (p=0.05) 0.75 1.92 0.17
Values are given as mean±standard deviation; Superscripts in lower case (a,b, etc.) represent DMRT (Duncan's Multiple Range Test) ranking within the
column; MC1=species of Chlorella, Scenedesmus, Chlorococcum, and Chroococcus; MC2=species of Phormidium, Anaba ena, Westiellopsis,
Fischerella,andSpirogyra
CD critical difference
Environ Sci Pollut Res
Discussion
Sewage sludge, a carbon-rich residual matter produced during
wastewater treatment, is extensively used as a source of mac-
ronutrients (N, P, etc.) and micronutrients (Zn, Cu, Mn, etc.) to
improve the soil fertility of agricultural lands, especially in the
reclamation of degraded lands (Antolin et al. 2005; Fernandez
et al. 2009; Motta and Maggiore 2013). A number of earlier
studies have demonstrated the positive effects of s ewage
sludge on crop productivity and yield on its land applications
(Warman and Termeer 2005; Fernandez et al. 2009;Mondal
et al. 2015). However, the excessive use of sewage sludge
may lead to the plant toxicity, accumulation of heavy metals,
increase in salinity, and detrimental effect on soil microbial
communities (Antolin et al. 2005;Mondaletal.2015). Earlier
studies also revealed the use of various types of wastewater
(treated and/or untreated), viz. rubber-processing industrial
wastewater (Owamah et al. 2014) and municipal wastewater
(Vivaldi et al. 2013), for fortification of agricultural lands, but
in this context, the microbiological contamination is a crucial
issue (Vivaldi et al. 2013). Yasmeen et al. (2014)demonstrat-
ed that biologically treated wastewater did not show any del-
eterious effect on Vigna radiata L. as compared with untreated
wastewater. Among various biological methods of wastewater
treatment (bacteria, fungi, algae, aquatic macrophytes, etc.),
microalgae have proved promising, since, in this process, the
sludge or biomass can be used as a nutrient-rich product, suit-
able for use in soil remediation and treated wastewater can be
used for various applications (Cabanelas et al. 2013;Renuka
et al. 2015). The use of microalgae is also advantageous in
terms of the production of nutrient-rich biomass, through se-
questration of valuable nutrients (N, P, micronutrients), which
can be exploited as a biofertilizer in crops. Despite the advan-
tages regarding the use of chemical fertilizers to increase crop
productivity; their continuous and long-term use may lead to
macro- and micronutrient deficiencies in soil (Zhang et al.
2010). In this context, the use of biofertilizers is emerging as
an economically viable and eco-friendly option (Bashan 1998;
Kaushik 1998). The integration of biofertilizers with inorganic
fertilizers in the package of practices for different crops can be
beneficial not only for the sustenance of crop production but
also for help in improving and restoring soil fertility and
health (Singh et al. 2011; Prasanna et al. 2012a, 2014).
Wheat is the one of major cereal crops, which utilizes a
large share of chemical fertilizers. Therefore, the use of
biofertilizers is a beneficial approach in terms of economic
viability and environmental sustainability. Cyanobacteria
have been recommended for wheat crop as a biofertilizer
(Karthikeyan et al. 2007; Nain et al. 2010; Prasanna et al.
2014). The colonization of cyanobacteria in wheat plant roots
was reported in earlier studies (Sood et al. 2011;Babuetal.
2015). In a hydroponics study, Babu et al. (2015)reportedan
increase in defense enzyme activity in root and 30–60 % in-
crease in plant fresh weight in wheat crop after 2 weeks of
culture. In a study by Maqubela et al. (2009). inoculation of
cyanobacterium
Nostoc le
d to 17–40 % increase in soil N.
Therefore, the inoculation of cyanobacteria can lead to a de-
crease in the use of chemical fertilizers. A useful intervention
can be the use of wastewaters rich in nutrients as low-cost
media for microalgal biomass generation.
In this study, microalgal consortia cultivated in sewage
wastewater were evaluated for their potential application as a
biofertilizer in wheat crop. Higher nutrient (N, P, and K) avail-
ability was obtained at mid crop stage in microalgal
consortium-inoculated treatments in comparison with non-
inoculated treatments and treatment with a full dose of fertil-
izer. It is well established that nitrogen is the most important
nutrient involved in plant growth and the use of 75 % N+full-
dose PK+MC2 (T5), leading to an increase of 8.91 % in the
available N in soil at mid crop stage.
Microbial activity in the rhizosphere determines the avail-
ability of nutrients to the plant and is used to assess the impact
of agronomic practices on soil vitality (Manjunath et al. 2011;
Gatica and Cytryn 2013). An increase in microbial activity
was recorded in terms of ARA, supported by the increase in
microbial biomass carbon of soil, in the microalgae/
cyanobacteria-inoculated treatments. Nitrogenase activity at
harvest stage revealed significantly highest ARA in T5
(75 % N+full-dose PK+MC2) treatment followed by T4
(75 % N+full-dose PK+MC1). In our study, the inoculated
consortium MC2 comprises cyanobacterial genera (species of
Anabaena, Westiellopsis, Fischerella), which are capable of
fixing atmospheric nitrogen, leading to higher ARA activity
and, hence, higher available nitrogen in soil at mid crop stage.
In the present study, T4 (75 % N+full-dose PK+MC1), which
includes unicellular forms, also showed an increase in the
ARA in soil samples that could be due to its ability to increase
the diazotrophic flora. However, the difference between the
available N of T4 and T1 (recommended dose NPK) was not
significant at mid and harvest stage, but T4 led to 17.5 %
higher nitrogen accumulation in grains.
An increase in soil available N was also observed on sew-
age sludge application to degraded soil of the Mediterranean
27
30
33
36
39
42
Full dose NPK 75%N + Full
dose PK
75%N + Full
dose PK +
carrier
75%N + Full
dose PK +
MC1
75%N + Full
dose PK +
MC2
1000 grain weight (g)
Treatment
Fig. 3 Effect of microalgal consortia on grain weight (1000-grain
weight) at harvest stage
Environ Sci Pollut Res
climate zone with barley crops (Antolin et al. 2005). The study
of Fernandez et al. (2009) revealed higher total N in soils
amended with composted and thermally dried sewage sludge,
compared with unamended and mineral-fertilized soils in a
semiarid Mediterranean agroecosystem. Similarly, Nain et al.
(2010) also reported higher ARA at mid crop stage and higher
values for available N at harvest stage in microalgae/
cyanobacteria-inoculated treatments in a pot experiment on
wheat crop. In the present study, sewage-grown microalgal
consortium-inoculated treatments also recorded higher avail-
able N at mid and harvest stage. T5 (MC2) exhibiting signif-
icantly higher available N at mid and harvest stage also
showed significantly higher N content in root and shoot at
mid crop stage. This also resulted in the higher uptake/
accumulation of N in the grains at harvest stage, which was
29 % higher as compared with control with a recommended
dose of NPK. Higher accumulation of N in barley grains was
also reported, in soil amended with sewage sludge, possibly
due to higher available N (Antolin et al. 2005). Similarly,
higher concentrations of N in straw and grains were recorded
in wheat plants grown in soil amended with sewage sludge
(Latare et al. 2014). In the present study, higher accumulation
of N in T5 might be because of higher N availability due to the
inoculation of nitrogen-fixing microalgal strains (MC2). Rana
et al. (2012) reported an increase in the accumulation of N
(24 %) in wheat grains in cyanobacterial and/or rhizobacterial
inoculations in comparison with the treatment with a full dose
of NPK. Nitrogen is known to influence the mobility and
uptake of micronutrients Zn and Fe, as it has been reported
that transporters for these micronutrients are greatly influ-
enced by the N nutrition status of the plant (Grotz and
Guerinot 2006).
Microbial biomass carbon represents an important index of
the microbial metabolic activities. In the present study,
microalgae/cyanobacteria-amended soil also resulted in
higher microbial biomass carbon at mid crop stage, compared
with uninoculated treatments/control. A similar increase in
MBC through inoculation of rhizobacteria and cyanobacteria
was also observed in earlier studies (Karthikeyan et al. 2007;
Nain et al. 2010; Prasanna et al. 2012a). In a pot experiment
conducted by Karthikeyan et al. (2007) on wheat crop, higher
microbial biomass carbon was obtained with the inoculation
of cyanobacterial consortium, as compared with individual
strains or recommended dose of fertilizer. In another study
on wheat crop, higher values for MBC were recorded in
cyanobacteria-inoculated treatments at mid crop stage as well
as harvest stage (Prasanna et al. 2012a). An increase in MBC
was also observed on sludge application to the agricultural
lands of different regions (Fernandez et al. 2009). Mondal
et al. (2015) reported a 92 % increase in MBC in sewage
sludge-treated field soil as compared with control. In the pres-
ent study, an increase of 31.8–67.0 % was recorded in MBC
values in microalgal consortium-inoculated treatments (T5
and T4) as compared with T1 control (full dose of fertilizer)
at mid crop stage. Further, these values were 3–4-fold higher
than other uninoculated treatments. Among the microalgal
consortium-inoculate d treatments, T4 (MC1-inoculated
treatment) recorded higher MBC as compared with MC 2-
inoculated treatment (T5). In soil as well as in aquatic envi-
ronment, microalgae are associated with bacteria (de-Bashan
et al. 2004; Lakaniemi et al. 2012). The a ssociation of
Chlorella spp. with bacteria is documented in earlier study
(de-Bashan et al. 2004; Lakaniemi et al. 2012). The
exopolysaccharides produced by microalgae provides food
to the bacterial partner, while bacterial partner produces plant
growth-stimulating hormones which enhance algal growth.
The sewage-grown microalgal biomass usually contains asso-
ciated bacteria (Su et al. 2011).andinthepresentstudy,
microalgal biomass was also f ound to harbor bacteria
(Renuka 2015) and might result in higher microbial biomass
carbon in T4.
Ph
osphorus is the second most important element involved
in plant growth and is next to the nitrogen in terms of require-
ments. The average P content in soil is about 0.05 % (w/w),
out of which only 0.01 % could be available to plants due to
the insoluble forms (Sharma et al. 2013). In India, approxi-
mately 98 % of the cropland is deficient in soil phosphorus,
due to the continuous cropping and low biological inputs
(Sharma et al. 2013). In the present study, inoculation of
microalgae/cyanobacteria (T4 and T5) resulted in an increase
in soil phosphorus at mid crop and harvest stages. This could
be due to their ability to mobilize insoluble forms of inorganic
phosphate present in soil (Hegazi et al. 2010). Cyanobacteria
are capable of mineralization of inorganic phosphorus into
soluble forms (Natesan and Shanmugasundaram 1989).
Cyanobacterial strains of Westiellopsis prolifica and
Anabaena variabilis were documented for mineral phosphate
solubilization through phthalic acid as a possible mode of P
solubilization; however, reports on green algae for improving
P availability from soil are rare. However, de-Bashan and
Bashan (2004) described the role of Chlorella spp. in nutrient
sequestration from wastewater, incorporating wastewater nu-
trients into algal biomass for their future use as a biofertilizer.
A study conducted by McColl (1975) suggested the possibil-
ity of e nzyma tic degradatio n of inorganic phosphorus by
green alga Chlor ella vulgaris. The addition of inorganic phos-
phorus and soil particles resulted in an increase in Chlorella
cells as well as available phosphorus in soil (McColl 1975).
The other reason for the increase in the nutrient availability
could be due to the presence of associated bacteria in sewage-
grown microalgal biomass, which can help in the mineraliza-
tion of insoluble nutrients. Available P at harvest stage also
recorded higher values in sewage-grown microalgae-inoculat-
ed treatments, and therefore, it is envisaged that microalgae
can help in the maintenance of nutritional status of soil.
Increased availability of P was also recorded with sewage
Environ Sci Pollut Res
sludge-amended soil over 3 years (Fernandez et al. 2009). and
higher concentrations of P in wheat straw and grain were
recorded with increased levels of sewage sludge in a pot ex-
periment with alluvial soil (Latare et al. 2014). In the present
study, in spite of the higher availability of P in the soil in
microalgae/cyanobacteria-inoculated treatments, no signifi-
cant differences were observed in the uptake and
accumulation of P as compared with the control with a full
dose of NPK. However, Rana et al. (2012)recordedanin-
crease of up to 54 % in P content of wheat grains with different
cyanobacterial and rhizobacterial inoculations.
Wheat is a K-demanding cereal crop, and uptake ranges
50–300kgha
−1
season
−1
(Zorb et al. 2014). Therefore,
lower/unbalanced K application may result in the depletion
of soil available K. Composted and thermally dried sewage
sludge application is reported to increase the available K in
soil in a semiarid Mediterranean agroecosystem (Fernandez
et al. 2009). Generally, sewage sludge contains a very low
concentration of K, as most of K in the wastewater readily
passed with the effluent (Warman and Termeer 2005).
Therefore, it can be envisaged that the greater available K in
sewage sludge-amended soil could be due to the higher avail-
ability of K because of increased microbial activity capable of
K solubilization. The key factors involved in the release of K
from the soil are the microbial population in the rhizosphere
capable of K solubilization and the effective uptake of K by
plant species (Zorb et al. 2014). In the present study,
microalgal consortium inoculation enhanced the available K
content in soil with highest values in MC2 at mid and harvest
stage compared with uninoculated treatments. MC2 inocula-
tion also resulted in higher accumulation of K in grains.
Rhizosphere bacteria play a major role in the availability of
K to the plants (Han et al. 2006; Parmar and Sindhu 2013).
The activity of K-solubilizing bacteria increases the availabil-
ity of nutrients to the plant, where polysaccharides play a
major role (Han et al. 2006; Sheng and He 2006).
Microalgal consortium inoculation may facilitate K solubili-
zation via the release of exopolysaccharides and/or support the
growth of K-solubilizing bacteria. In this study, an increase in
the soil available N in the microalgal consortium-inoculated
treatments with highest values in T5 (75 % N+full-dose PK+
MC2) also resulted in the higher accumulation of potassium in
grains. A total of 25.0 and 28.5 % higher K accumulation in
grains was noticed in T4 (75 % N+full-dose PK+MC1) and
T5 in comparison with the control with a full dose of fertilizer.
The concentration of potassium influences the wheat grain
protein through the enhanced translocation of nitrogenous
compounds from shoot and also stabilizes the proteins for
storage (Zorb et al. 2014).
Potassium (K) deficiency in the cereal crops can restrict the
utilization of N by the plant and may result in the nitrate
leaching (Zorb et al. 2014). Potassium uptake potential is
known to vary among various plant species and is dependent
upon various factors, viz. root structure, density, and length
(Nieves-Cordones et al. 2014; Wigoda et al. 2014;Zorbetal.
2014). A positive correlation between root hair length/density
and K content was reported in various crops (Junk 2001;
Hogh-Jense and Pedersen 2003). In our study, a strong posi-
tive correlation (r=0.95) was observed between K accumula-
tion in t he grain and root length, whic h substantiates the
higher values observed in the roots and grains. The compari-
son of values recorded for the available K in soil at mid and
harvest stages of crop revealed lower differences in the treat-
ments involving microalgal consortium-inoculated treat-
ments. This illustrates a greater increase in the residual level
of K at harvest stage, which can beneficial for the succeeding
crop. Therefore, the utilization of such microalgal consortia in
agricultural practices may help in the maintenance of soil nu-
tritional status.
Soil nutrient availability played a major role in crop pro-
ductivity and yield. The enhancement of biological activity
and chemical characteristics of soil also led to the increase in
crop yield and quality of grains (Hegazi et al. 2010). In the
present study, a higher number of tillers and root length in
microalgal consortium-inoculated treatments were observed
at mid crop stage in comparison with the treatment with a full
dose of fertilizer. An increase in crop yield parameters at har-
vest stage, viz. plant dry weight, spike weight, number of
spikes, and grain weight, was also observed in treatments
inoculated with microalgal consortia and an ultimate increase
in harvest index. Crop biometrical characteristics are directly
related to the nutritional status of soil and plant. In our study,
plant biometrical parameters (crop yield) were positively cor-
related with plant and soil nutrients. Grain weight and nutrient
level were directly correlated with the amount of nutrients (N,
P, and K) present in soil and plant. Similar observations for
wheat grain yield were observed by Rana et al. (2012). An
increase in plant productivity through the cyanobacterial inoc-
ulation was also reported in earlier studies, as these organisms
(cyanobacteria) secrete plant hormones and other substances
which increase plant growth and enhance the availability of
nutrients in the soil (Nain et al. 2010; Manjunath et al. 2011;
Prasanna et al. 2012a
;Ranaetal.20
12; Lavakush et al. 2014).
Manjunath et al. (2011) also recorded higher crop yield and
plant growth characteristics, viz. shoot and root length and
biomass, with inoculation of Anabaena strains individually
and/or with proteobacteria, which increases the availability
of nutrients in soil and ultimately leads to the higher plant
growth and yield. Sludge application in alluvial soil has been
reported to increase grain weight and yield in wheat crop, as
compared to the use of the recommended dose of fertilizers
(Latare et al. 2014). The direct use of sludge and/or wastewa-
ter for fortification of cropland to increase the crop yield has
been reported (Antolin et al. 2005; Latare et al. 2014;
Owamah et al. 2014). which is directly related to the increased
availability of nutrients as a result of enhanced microbial
Environ Sci Pollut Res
activity (Kizilkaya and Bayrakh 2005; Motta and Maggiore
2013). In the present study, an increase in crop yield, grain
weight, and nutritional status of grain (particularly N) in MC2-
inoculated treatments compared with control (full dose of fer-
tilizer) can be due to the presence of cyanobacterial
genera, capable of nitrogen fixation and production of
exopolysaccharides, and hence increased availability of nutri-
ents. However, the highest plant dry weight and the number of
tillers were observed at mid crop stage in MC1 (microalgal
consortium of unicellular strains)-inoculated treatment (T4).
The comparatively higher plant growth attributes in T4 could
be due to the release of plant hormones and/or metabolites or
copious production of exopolysaccharides by unicellular
green algae/cyanobacteria, as also reported in earlier studies
on lettuce plant (Faheed and Fattah 2008). Faheed and Fattah
(2008) evaluated the potential of C. vulgaris as a biofertilizer
on growth parameters and metabolic aspects of lettuce plant
and found a significant increase in the growth of lettuce plants
and uptake of nutrients from sterilized autoclaved media on
microalgal treatment. They also recorded significantly higher
fresh and dry weight of seedlings (Faheed and Fattah 2008).
which is similar to our observations in the present study.
Conclusions
The present investigation illustrated the utility of sewage-grown
biomass of two microalgal consortia of filamentous and unicel-
lular strains on growth and yield of wheat crop. Both the con-
sortia enhanced the crop productivity and yield, as compared
with the recommended dose of NPK fertilizers. Enhanced mi-
crobial activity as well as greater pool of available nutrients at
mid crop stage were observed in microalgal consortium-
inoculated treatments, which may be responsible for the signif-
icantly higher grain yield and weight. The study highlighted the
promise of sewage-grown microalgal consortia as a potential,
low-cost, and sustainable biofertilizer for wheat crop. Field lev-
el evaluation of such sewage-grown biomass as nutrient sup-
plements in integrated nutrient management practices can help
in the evalu ation of their agronomic efficiency.
Acknowledgments The first author is thankful to the University Grants
Commission, New Delhi, for her fellowship. All the authors are thankful
to the Department of Botany, Panjab University, Chandigarh, and to the
Division of Microbiology, Division of Agronomy, and National
Phytotron Facility, ICAR-Indian Agricultural Research Institute, New
Delhi, for providing t he resea rch f acilities to carry out the pres ent
investigation.
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