Wastewater grown microalgal biomass as inoculants for improving micronutrient availability in wheat

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Rhizosphere
journal homepage: www.elsevier.com/locate/rhisph
Wastewater grown microalgal biomass as inoculants for improving
micronutrient availability in wheat
Nirmal Renukaa,e, Radha Prasannab,⁎, Anjuli Soodb, Radhika Bansald, Ngangom Bidyaranib,
Rajendra Singhc, Yashbir S. Shivayd, Lata Nainb, Amrik S. Ahluwaliaa
a Department of Botany, Panjab University, Chandigarh 160014, India
b Division of Microbiology, ICAR-Indian Agricultural Research Institute, New Delhi 110012, India
c National Phytotron Facility, ICAR-Indian Agricultural Research Institute, New Delhi 110012, India
d Division of Agronomy, ICAR-Indian Agricultural Research Institute, New Delhi 110012, India
e Institute for Water and Wastewater Technology, Durban University of Technology, P.O. Box 1334, Durban 4000, South Africa
A R T I C L E I N F O
Keywords:
Sewage
Microalgal consortia
Micronutrients
Biofortification
Wheat
A B S T R A C T
An investigation was undertaken to evaluate the potential of two sewage grown microalgal formulations
(consortia of native microalgae mixed with vermiculite: compost as carrier) in enhancing the soil micronutrient
availability and uptake in wheat crop. Significantly higher available zinc (Zn), iron (Fe), copper (Cu) and
manganese (Mn) content were recorded in soil samples from treatments belonging to microalgal consortia
inoculation, as compared to uninoculated treatments, at both mid and harvest stage of wheat crop. A significant
enhancement of 35.1 - 51% in organic carbon content was recorded in microalgal consortia inoculated
treatments over control. Highest values were observed in treatment T5 (75% N + full dose PK + formulation
with MC2, comprising native filamentous microalgae (Phormidium, Anabaena, Westiellopsis, Fischerella,
Spirogyra). The treatment T4 (75% N + full dose PK + MC1formulation (comprising unicellular green algae
-Chlorella, Scenedesmus, Chlorococcum, Chroococcus)showed significantly higher values of dehydrogenase
activity. The plants from treatment T5 (75% N + full dose PK + formulation with MC2) recorded 53% higher
leaf chlorophyll, as compared to T1 (recommended dose of fertilizer) at mid-crop stage. Microalgal consortia
inoculated treatments also showed 37.3 - 48.0% increase in grain yield with significantly higher micronutrient
(Zn, Fe, Cu and Mn) content in grains, as compared to control. A strong positive correlation was recorded
between the availability of micronutrients in soil at mid-crop and grain yield. The present study highlighted the
promise of wastewater grown microalgal consortia for improving soil micronutrient availability, micronutrient
enrichment of wheat grains and enhancing crop productivity.
1. Introduction
Microalgae are considered as an excellent feedstock for a wide range
of products - bioactive compounds, biofuels and biofertilizer and their
diverse applications in wastewater treatment, carbon sequestration etc.
(Gupta et al., 2013; Renuka et al., 2015). Although there are many
studies illustrating their role in wastewater treatment and biomass
production (Chinnasamy et al., 2010; Guldhe et al., 2017), published
literature on the application of wastewater grown microalgae biomass
as biofertilizer is scanty (Wuang et al., 2016). Micronutrients play an
important role in plant metabolism and their availability in soil directly
affects the physiological functioning of plant and quality of plant-
products (Prasanna et al., 2013; Rana et al., 2012). According to recent
estimates, about two billion people in the world are affected by
micronutrient deficiency, with a higher prevalence in developing
regions like Southeast Asia and sub-Saharan Africa (Ramakrishnan,
2002). Wheat is among the most important staple food crops among
cereals, which contributes towards 50% of the total energy intake in the
Indian sub-continent (Akhtar et al., 2011). Recent agricultural practices
are mainly focused on achieving higher agronomic yields, and bioforti-
fication plays a secondary role, particularly in relation to improving the
nutritional status of edible parts of the crop. Further, the use of non-
judicious and excessive application of chemical fertilizers to meet
global demands has resulted in the decreased accumulation of Zn, Fe,
Cu and Mn in cereal grains, which are the critical micronutrients
sources in the human diet (Zhang et al., 2012).
In this context, the use of microalgae is an eco-friendly and
economically viable approach to enhance the crop quality and produc-
http://dx.doi.org/10.1016/j.rhisph.2017.04.005
Received in revised form 6 April 2017; Accepted 6 April 2017
⁎ Corresponding author.
E-mail address: radhapr@gmail.com (R. Prasanna).
Rhizosphere 3 (2017) 150–159
Available online 08 April 2017
2452-2198/ © 2017 Elsevier B.V. All rights reserved.
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tivity and also help to maintain the soil fertility (Karthikeyan et al.,
2007, Perez-Montano et al., 2014). However, mass production of
microalgae also requires massive quantities of nutrient inputs
(Borowitzka and Moheimani, 2010, Markou et al., 2014). It has been
estimated that commercial cultivation of microalgae requires approxi-
mately three times higher fertilizer inputs than terrestrial plants
(Markou et al., 2014), which questions their economic and environ-
mental impact. A valuable resource is wastewaters; they contain a
majority of valuable nutrients needed for the proliferation and mass
multiplication of algae (Chinnasamy et al., 2010; Guldhe et al., 2017).
The use of wastewater nutrients as raw resource and/or in the form of
sludge is a low cost process attracting a lot of attention (Calleja-
Cervantes et al., 2017). As these wastewaters also harbor harmful
bacteria and are rich in heavy metals, direct application of wastewater
or in the form of sludge to the food crops can lead to pathogenic
bacteria in the vegetative/edible parts (Vivaldi et al., 2013), besides
accumulation of heavy metals (Yang et al., 2017). Direct application of
wastewater also increases the bacterial contamination in the soil,
leading to a decrease in water retention, soil porosity and hydraulic
conductivity (Aiello et al., 2007). Other problems related to the use of
sludge are the presence of chemical flocculants being used during
wastewater treatment, which decreases the mineralization of nutrients
(Warman and Termeer, 2005).
Therefore, the use of wastewater grown microalgae biomass for
fortification of agricultural soils, vis-a-vis sludge, could be a beneficial
approach. Wastewaters contain a majority of nutrients required for
microalgae cultivation (Guldhe et al., 2017), and the heavy metals
present in wastewater in trace amounts act as a source of micronu-
trients for the growth of microalgae/cyanobacteria(Wang et al., 2010).
Therefore, microalgae play a double role in wastewater treatment -
through scavenging the available macro/micronutrients/heavy metals
and production of useful biomass which can be utilized as a source of
biofertilizer (Markou et al., 2014; Wuang et al., 2016). The growth of
algae in these wastewaters is advantageous, as it reduces the bacterial
contamination, including human pathogens, because of the alkaline
conditions (Ansa et al., 2012; da Silva Ferreira et al., 2017). Reports are
available on the utilization of microalgae/cyanobacteria as biofertilizer,
plant growth promoters and bio-control agents for various crops
(Chaudhary et al., 2012). These organisms are also reported to increase
the yield and micronutrient concentration in wheat crop (Prasanna
et al., 2013; Rana et al., 2012). Sewage grown microalgal biomass can
be an economically viable option for the fortification of soils used in the
cultivationof cereals. In our previous study, sewage grown microalgal
consortia proved promising for use as N/P/K fertilizer with 25% N
savings in wheat (Renuka et al., 2016). Therefore, in this study, the
potential of sewage grown microalgal consortia of filamentous and
unicellular strains (both cyanobacteria and green algae) were evaluated
as inoculants in wheat, for micronutrient enrichment in grains and
facilitating enhanced micronutrient (Zn, Fe, Cu and Mn) dynamics in
soil and plant.
2. Materials and methods
2.1. Microalgal consortia used and their growth
Two microalgal consortia made up of cyanobacteria (Cyanophyta)
and green algae (Chlorophyta),were developed and analyzed for their
potential in wastewater remediation, and as inoculants in wheat crop in
our earlier studies (Renuka et al., 2013a, 2013b). These microalgal
consortia viz. MC1 comprised native unicellular strains of sewage
[species of Chlorella (Chlorophyta), Scenedesmus (Chlorophyta), Chlor-
ococcum (Chlorophyta), Chroococcus (Cyanophyta)] and MC2, made up
of native filamentous strains isolated from sewage wastewater [species
of Phormidium (Cyanophyta), Anabaena (Cyanophyta), Westiellopsis
(Cyanophyta), Fischerella (Cyanophyta), Spirogyra (Chlorophyta)]. De-
tails regarding taxonomic description for each strain of microalgal
consortia, development of the consortia after selection and their
screening is given in our earlier publication (Renuka et al., 2013b).
For consortia development, equal amounts of each microalgal strains
(0.1–0.2 µg mL−1 on chlorophyll basis) were mixed and allowed to
grow in modified Bold's and Basal Medium (Starr and Zeikus 1993)
under outdoor conditions in a polyhouse. for acclimatization. Twenty
days old microalgal consortia were harvested, washed with distilled
water to remove the adhering material,and used as inoculum in
wastewater.
Sewage wastewater was collected in the plastic containers of 15 L
capacity from the channel at ICAR-Indian Agricultural Research
Institute, New Delhi, whose characteristics are given in
Supplementary Table 1. After primary settling, the sewage wastewater
was filtered through muslin cloth, and used as the growth medium for
microalgae cultivation.Microalgae cultivation was carried out under
outer door conditions (at atmospheric night/day temperature and light
intensity ranging 16–32 °C and 350–1420 μmol photons m−2 s−1 re-
spectively)in a polyhouse in plastic tubs of 30 L capacity with working
volume of 15 L. Microalgal consortia grown in sewage wastewater were
harvested on 6th day of growth, air dried and used for further
experimentation.
2.2. Experimental design
An experiment was conducted in pots of 8” size, containing 6 kg of
sterile soil, with wheat variety HD2967 under the controlled environ-
ment chamber of the National Phytotron Facility, IARI, New Delhi,
which simulated optimal conditions (24±2 °C and 20±2 °C; day and
night temperature respectively) for the growth of wheat crop. The soil
type used was sandy loam, Inceptisol (Udic Ustocrept), with the initial
physicochemical properties, as given in Supplementary Table 2. The dry
biomass of microalgal consortia (20 µg chlorophyll g−1 carrier) was
added to vermiculite: compost (1:1), as a carrier for preparing the
formulations, based on earlier published reports (Prasanna et al.,
2013).The characteristics of the carrier are as given in Chaudhary
et al. (2012).The formulations were stored at room temperature and
maintained at 60% water holding capacity. The characteristics of
microalgal consortia formulations with carrier are given as
Supplementary Table 2. Fifty grams of each formulation was mixed
with 6 kg soil in the respective pots. The macro and micronutrient
composition of the microalgal formulations (with carrier) of MC1 and
MC2 are: N (7.5% and 12.9%), P (1.49% and 1.52%), K (3.56% and
4.60%), Zn (3.38 and 7.57 µg g−1), Fe (195.6 and 145.0 µg g−1), Cu
(0.092 and 0.080 µg g−1), Mn (22.8 and 24.4 µg g−1) respectively. In
this study, a total of five treatments were taken viz. T1(Recommended
dose of fertilizer N: P: K − 120: 60: 60 kg/ha), T2(75% N + full dose
PK), T3 (75% N+ full dose PK + carrier), T4 (75% N+ full dose PK +
MC1) and T5 (75% N + full dose PK + MC2). Irrigation of soil in pots
was done regularly to maintain the 60% water holding capacity. Seeds
were soaked and kept in dark conditions for 24 h. There were six
replicate pots for each treatment and five seeds of wheat were sown in
each pot with equal spacing. 50% of N was supplemented at the time of
sowing in T1 control and other treatments respectively, while the
remaining dose was supplied at the tillering stage. The microalgal
consortia are expected to provide 20–30 kg N/ha and therefore used
along with 75% N and recommended doses of P and K fertilizer (Renuka
et al., 2016).
Plant and soil samples were collected at mid (60 DAS - days after
sowing) and harvest stage. Three replicates pots were used for the
analyses at mid-crop stage, while the remaining three were utilized at
harvest stage. From each pot, a minimum of three plants were taken for
the analyses of plant related parameters. Soil cores from the root
region/rhizosphere were collected in triplicate using auger from each
pot for the assessment of microbiological parameters.
N. Renuka et al. Rhizosphere 3 (2017) 150–159
151
2.3. Analyses of soil parameters
Soil samples were collected from the root rhizosphere cores
(0–15 cm depth). Micronutrient analyses (Fe, Mn, Zn and Cu) in soil
samples were carried out using DTPA (diethylene-triamine-pentaacetic
acid)- extraction and an Atomic Absorption Spectrophotometer at the
most sensitive wavelengths −248.7 nm, 324.6, 213.7, and 279.5 nm
for Fe, Cu, Zn and Mn respectively (Lindsay and Norvell, 1978).
Estimation of soil chlorophyll was carried out by the method described
by Nayak et al. (2004), using acetone and dimethyl sulphoxide (DMSO)
in 1:1 ratio as extractant. Organic carbon was measured using
dichromate oxidation method as described by Jackson (2005). Dehy-
drogenase(EC 1.1.1.) activity in soil samples was assayed after extrac-
tion with methanol using triphenyl tetrazolium chloride (3%). Absor-
bance was measured at 485 nm and values were expressed as μg
Triphenyl Formazon (TPF) g−1 soil d−1(Ameloot et al., 2014). Mea-
surement of ethylene (index of nitrogenase activity) was carried out
using the acetylene reducing assay (ARA), using a Gas Chromatograph
(Bruker 450GC). Quantification was done using ethylene (1000 ppm;
Sigma Gases and Services, New Delhi, India) as standard and similar
vials with equivalent amount of water served as control. The column
temperature was maintained at 70°C, while injector and detector
temperature was maintained at 150 °C and 250 °C respectively. The
amount of nitrogen fixed was calculated by multiplying ARA (Acetylene
reducing activity) values with factor 3 and expressed as nmol N fixed
g−1 soil h−1 (Berrendero et. al., 2016).
Bacterial load in soil samples was evaluated according to the
method described by Brown and Smith (2014). XLD agar and SS agar
(Salmonella Shigella Agar, HiMedia, India) were used as media for the
enumeration of Salmonella spp., Shigella spp. and Escherichia coli, while,
nutrient agar (HiMedia, India) was used for the estimation of total
bacterial counts.
2.4. Analyses of plant parameters
For the analyses of micronutrients (Zn, Fe, Cu and Mn) in wheat
plants at mid-crop and harvest, the samples (grain, shoot and root
separately) were finely ground and digested. Analyses for micronutrient
concentration in the digests were done using atomic absorption
spectrophotometer (Lindsay and Norvell, 1978). Leaf chlorophyll
content was measured by the method given by Hiscox and Israelstam
(1979); and equations described by Arnon (1949). Plant biometrical
parameters, including number of spikes per pot and grain yield per pot
(expressed as g pot−1) were measured at the harvest stage of crop.
2.5. Statistical analyses
The analyses of results were carried out using the Statistical package
for SocialSciences (SPSS Version 16.0). One-way analysis of variance
(ANOVA) was used to evaluate the differences among the treatments.
The triplicate sets of data were evaluated in accordance with the
experimental design (Completely Randomized Design). The compar-
isons between the different means were made using post hoc least
significant differences (LSD) calculated at P level of 0.05 (5%), and
represented as C.D. (Critical Differences) values in Tables. SD (Standard
deviation) values are depicted in the graphs as error bars. DMRT
Fig. 1. (a-b) Percent change in the micronutrient content of soil, as influenced by microalgal inoculation, as compared to T1 control (Recommended dose of fertilizer) (a) at mid-crop
stage (b) at harvest stage of wheat crop.
N. Renuka et al. Rhizosphere 3 (2017) 150–159
152
(Duncan's Multiple Range Test) analyses are depicted as superscripts in
table and graphs, with ‘a’ representing highest values. Correlation
coefficients were calculated by using Microsoft Excel package and
analyzed for their significance using Pearson's tables.
3. Results
3.1. Micronutrient content in soil and plant samples
Inoculation of sewage grown microalgal consortia increased the
availability of micronutrients in soil and also showed improvement in
plant nutritional characteristics (Figs. 1–3). Percent change in micro-
nutrient concentration in soil and plant samples in different treatments
was calculated in comparison to the control treatment, supplied with
recommended doses of NPK (Figs. 1–3). Analyses of Zn content in soil
samples at mid-crop stage revealed the highest increase of 20.8% in Zn
content in T5 (75% N + full dose PK + MC2), followed by T4 (75% N
+ full dose PK + MC1) (18.8%) as compared to T1 (Recommended
dose of NPK) (Fig. 1a). However, at harvest stage, highest increase in Zn
content in soil was observed in treatment T4 (26.63%), followed by T5
(24.26%). In uninoculated treatments, Zn content was not significantly
different in shoot and root at mid-crop stage. Treatment T4 showed the
highest increase of 75.9% and 134.7% in Zn content at mid-crop in
shoot and root respectively, as compared to control T1 (Recommended
dose of NPK) (Fig. 2a-b). Sewage grown microalgal consortia inocula-
tion led to 10–12% greater accumulation of Zn in grains, with highest
value in T5 (Fig. 2a). At harvest stage, highest increase in shoot Zn
content (27.2%) was recorded in T4, while T5 showed highest increase
in root Zn content (Fig. 2b-c).
Highest increase in soil Fe content (61.3%) was exhibited by T4
followed by T5 (30.9%) at mid-crop stage (Fig. 1a). A similar trend for
Fe content in soil samples was also observed at harvest stage. T4
showed highest increase of 77.8% in soil Fe content followed by T5
(Fig. 1b). Microalgal consortia inoculated treatments also revealed
higher Fe content in plant samples as compared to uninoculated
treatments. Results revealed that the highest increase in shoot and root
Fe content was recorded in T5 (75% N + full dose PK + MC2) at mid-
crop stage (Fig. 2a-b). Microalgal consortia inoculated treatments
showed 24–42% enhancement in Zn accumulation in grains. T5
recorded highest Zn accumulation in grains and showed 41.6% increase
in Zn content as compared to T1 (Fig. 3a). Fe content in shoot and root
showed a similar trend at harvest stage with significantly higher values
in T5. T5 exhibited highest increase of 59.5% and 102.9% in Zn content
in shoot and root at harvest stage respectively (Fig. 3b-c).
Cu content in soil samples revealed highest increase of 36.9% and
92.6% in T5 at mid and harvest crop stage respectively (Fig. 1a-b).
Similarly, T5 also showed highest increase in Cu content in shoot
(54.2%) and root (28.9%) at mid-crop stage respectively. At harvest
stage, higher accumulation of Cu in grains was observed in T5 (54.8%)
followed by T4 (48.2%) compared to T1 (Fig. 3a). However, there was
no significant change in shoot Cu content at harvest stage in microalgal
consortia inoculated treatments, compared to T1 control (Recom-
mended dose of fertilizer). A decrease of 8% in Cu content was recorded
in T5, compared to T1 control (Fig. 2b). Microalgal consortia inoculated
treatments showed 10–15% higher Cu content in root, as compared to
T1 control (Fig. 3c).
Fig. 2. (a-b) Evaluation of microalgal consortia, in terms of percent change in content of various micronutrients in wheat plants, in comparison with T1 control (Recommended dose of
fertilizer) at mid-crop stage (a) shoot (b) root.
N. Renuka et al. Rhizosphere 3 (2017) 150–159
153
An increase in Mn content in soil was observed in microalgae
inoculated treatments at mid and harvest crop stage (Fig. 1a-b). T5
exhibited highest increase in Mn content at mid-crop (25.9%) and
harvest stage (77.3%), compared to T1. Despite the higher accumula-
tion of Mn content in shoot and root in T4 at mid-crop stage, as
compared to T5, Mn content in grains was more in T5; 72.9% increase
in grain Mn content compared to T1 control. However, a decrease in Mn
content in root and shoot was observed in microalgal consortia
inoculated treatments as compared to T1 control (Recommended dose
of fertilizer) at harvest stage.
These results revealed that microalgal consortia inoculation led to
higher accumulation of micronutrients (Zn, Fe, Cu and Mn) in grains, as
compared to control (Recommended dose of fertilizer) with highest
values in T5 (75% N + full dose PK + MC2).
3.2. Soil microbiological parameters
Soil chlorophyll values of 4.43 and 4.97 mg g−1 were observed in
samples from microalgal consortia inoculated treatments T4 (75% N +
full dose PK + MC1) and T5 (75% N + full dose PK + MC2)
respectively, which were significantly higher than uninoculated treat-
ments (Table 1). Significantly higher values for nitrogen fixation were
recorded in T5 (17.59 nmol N fixed g−1 soil h−1), followed by T4
(14.42 nmol N fixed g−1 soil h−1) (Table 1). Inoculation of sewage
grown microalgae biomass led to 1.8 and 2.2-folds increase in nitrogen
fixation potential, as compared to T1 (Recommended dose of fertilizer).
An increase of 35.1- 51.3% in organic carbon content was recorded
in T4 and T5respectively as compared to T1 control (Table 1). Micro-
algal consortia inoculated treatments also revealed higher dehydrogen-
Fig. 3. (a-b) Influence of microalgal consortia application on micronutrient content in plant, represented as percent change, compared to T1 control (Recommended dose of fertilizer) at
harvest stage (a) grain (b) shoot (c) root.
N. Renuka et al. Rhizosphere 3 (2017) 150–159
154
ase activity as compared to uninoculated treatments, with highest value
of 110.53 µg g−1d−1 in T4 (75% N + full dose PK + MC1) (Table 1).
T4 (75% N + full dose PK + MC1) exhibited a higher bacterial load, as
compared to other treatments (Supplementary Table 3).
3.3. Plant parameters
Leaf chlorophyll at mid-crop stage revealed significantly higher
values in T5 (75% N + full dose PK + MC2) as compared to other
treatments (Fig. 4a). At harvest stage, more number of spikes per pot
were observed in microalgal consortia inoculated treatments with
highest value in T5 (Fig. 4b). An increase of 48 and 37.33% in total
grain yield (g pot−1) was recorded in T4 (75% N + full dose PK +
MC1) and T5 (75% N + full dose PK + MC2) respectively compared to
T1 control (Recommended dose of fertilizer) (Fig. 4c).
3.4. Correlation between plant and soil micronutrient characteristics and
yield parameters
The amount of N fixed was positively correlated with micronutrients
Zn (r = 0.89), Fe (r = 0.71), Cu (r = 0.76) and Mn (r = 0.87) in soil
samples at mid-crop stage. A positive correlation was recorded in soil
organic carbon and Zn (r = 0.91), Fe (r = 0.72), Cu (r = 0.79) and Mn
(r = 0.84) content in soil samples. Plant yield was directly related to
the soil micronutrients (mainly Zn and Fe) at mid-crop stage. A strong
positive correlation was exhibited between number of spikes and grain
yield (g pot−1), with soil Zn (r = 0.90, 0.80) and Fe content (r = 0.91,
0.87)at mid-crop.
4. Discussion
Imbalanced use of chemical fertilizers, along with reduced applica-
tion of recycled crop residues has led to the deficiency of macro and
micronutrients and impairment of the physical, chemical and biological
properties of soil (Geisseler and Scow, 2014; Li et al., 2017; Murase
et al., 2015). Sewage wastewater is a rich source of macro and
micronutrients, which can be used for fertigation of crops, particularly
cereals that are heavy nutrient feeders. However, the direct use of
sewage wastewater and/or sludge for the fortification of crops may lead
to enrichment of pathogenic flora and accumulation of toxic heavy
metals in soil and plant (Calleja-Cervantes al., 2017; Yang et al., 2017).
A beneficial and rational approach involves the utilization of micro-
algae/cyanobacteria, as they can remediate wastewater by growth,
scavenge and incorporate valuable nutrients into their biomass (Luo
et al., 2016; Markou et al., 2014). The use of microalgae/cyanobacteria
is desirable, as they not only provide incorporated micronutrients to the
growing crop, but also provide plant growth promoting substances,and
reduce the proliferation of harmful/ pathogenic bacteria (human and
plant pathogens), as a result of the alkaline oxygenated conditions
created as a result of their growth (Chaudhary et al., 2012; Prasanna
et al., 2013).
Wheat is one of the first domesticated cereals, and the most
important food grain source for human consumption mainly in
Europe, West Asia and North Africa. It is grown on the largest land
area, more than any other commercial crop and continues to be the
largest consumer of chemical fertilizers. Wheat is the dominant staple
food in many micronutrient-deficient regions (Cakmak et al., 2010)and
agronomic biofortification along with molecular breeding approaches
are being deployed to tackle malnutrition globally (Velu et al., 2014).
Zn deficiency is listed as a major risk factor for human health; according
to a WHO (World Health Organization) report, Zn deficiency is among
the most important risk factors responsible for illnesses and diseases in
developing countries (IFPRI (International Food Policy Research
Institute), 2016). Fe and Mn deficiency leads to the impairment of
physical and mental growth; and affects more than 47% of children
globally. Biofortification of wheat through the application of cyano-
bacteria/ microalgae is an important intervention, which not only
improves soil fertility, along with the improvement in yield, but also is
able to fortify micronutrients in plant tissues, including grains (Adak
et al., 2016; Rai et al., 2000). In our previous study, wastewater
cultivated microalgae, developed as consortia proved promising in
macronutrient (N/P/K) fortification with 25% N savings in wheat
(Renuka et al., 2016).
4.1. Effect of sewage grown microalgal consortia on micronutrient content
in soil and plant samples
In the present study, sewage grown microalgal consortia were
evaluated for their biofortification potential in wheat crop. Utilization
of sewage grown microalgal consortia was found to increase the
available micronutrient content (Zn, Fe, Cu, Zn) in soil and plant parts
at both mid-crop and harvest stage. An increase of 23 – 26.2% and 32 –
36% was recorded in Zn content of soil at mid-crop and harvest stage
respectively, as compared to application of recommended doses of
chemical fertilizers. However, no significant differences in the available
Zn content of soil samples at mid-crop or harvest stage among the
treatments inoculated with microalgal consortia were observed. This
illustrates the sufficient availability of Zn for the succeeding crop after
wheat. An increase of 10 – 11% in Zn content in wheat grains was also
observed with microalgal consortia inoculated treatments, in compar-
ison to control treatment.
The uptake of Zn and Fe is directly related to the nitrogen
nutritional status of the plant. The expression of transporters for Zn
and Fe is highly dependent on the plant nitrogen nutritional status
(Grotz and Guerinot, 2006; Guo et al., 2014). Xue et al. (2012)
suggested that optimal nitrogen supplementation and management in
wheat crop can lead to better shoot Zn accumulation and mobilization
to the grains. They achieved 30–72% increase in grain Zn accumulation
by increasing nitrogen supply to optimum levels. Earlier studies also
suggested that microalgae/cyanobacteria application to the crop can
Table 1
Analyses of soil microbiological parameters in different treatments at mid-crop stage.
Treatment Soil chlorophyll Nitrogen fixing potential Organic carbon Dehydrogenase activity
(mg g-1) (nmol N fixed g-1 soil h-1) (%) (µg g-1 d-1)
T1 (Recommended dose NPK) 1.01± 0.02e 8.06±0.20c 1.11± 0.21c 57.73± 6.83c
T2 (75% N + Full dose PK) 2.27± 0.30d 8.33±0.57c 1.01± 0.07c 54.33± 8.85c
T3 (75% N + Full dose PK + carrier) 2.74± 0.05c 8.39±0.45c 1.12± 0.15c 59.16± 10.40c
T4 (75% N + Full dose PK + MC1) 4.43± 0.13b 14.42± 0.98b 1.50± 0.16b 110.53± 11.53a
T5 (75% N + Full dose PK + MC2) 4.97± 0.04a 17.59± 1.29a 1.68± 0.04a 82.53± 4.70b
SEM 0.086 0.461 0.061 5.088
CD (p=0.05) 0.24 1.28 0.17 14.10
Values are given as mean (n=3)± Standard Deviation (S.D); SEM (Standard Error Mean); CD (Critical Difference); MC1 - species of Chlorella, Scenedesmus, Chlorococcum, Chroococcus);
MC2 - species of Phormidium, Anabaena, Westiellopsis, Fischerella, Spirogyra); Superscripts a,b,c etc. represent DMRT (Duncan's Multiple Range Test) ranking in different treatments for
respective parameter, where ‘a’ represents the highest value and dissimilar superscripts represent significantly different values.
N. Renuka et al. Rhizosphere 3 (2017) 150–159
155
replace 25–50% N requirement in agronomic practices (Prasanna et al.,
2013). In our study, 75% N supplementation to the growing crop had
no deleterious effects on the micronutrient uptake (especially Zn and
Fe) by the plant and their accumulation in grains. This can be due to
sufficient availability and supply of nitrogen to the growing crop, by the
nitrogen-fixing heterocystous filamentous strains (species of Anabaena,
Westiellopsis, Fischerella) of MC2 consortium in T5. In our previous
study, sewage grown microalgal consortia showed an increase in the
available nitrogen in the soil (Renuka et al., 2016). Microalgal growth
can also enhance the growth of rhizosphere bacteria, due to the
secretion of exopolysaccharides and other exudates (Kapustka and
DuBois, 1987). Cell extracts of some blue green algae and diatoms
exert stimulatory effect on the growth of siderophore producing
bacterium (Amin et al., 2009). They suggested that the photolysis of
iron siderophore stimulated Fe assimilation in aquatic environment
promotes algae-bacteria mutualism, wherein microalgae release organ-
ic molecules that are utilized by bacteria for growth. Such mutualistic
association between algae and siderophore producing bacteria play
imperative roles in the availability and utilization of Fe by these
organisms (Amin et al., 2009). Wilhelm et al. (1996) reported that
cyanobacteria produce and utilize iron chelators called siderophores in
low Fe and nitrogen (N) conditions, creating a competitive advantage
over other algae in freshwater lakes, but their function in aquatic or
terrestrial iron cycles remains less clear. Reports of siderophore
production by eukaryotic algae are even rarer, although there are
reports of macromolecular algal exudates involved in iron assimilation
(Fuse et al., 1993).
Further, phytosiderophore production by wheat plant root exudates
Fig. 4. (a) Comparative analyses of leaf chlorophyll content in different treatments at mid-crop stage of wheat crop (b-c) Plant yield parameters at harvest stage (b) Number of spikes per
pot (c) Grain yield per pot.
N. Renuka et al. Rhizosphere 3 (2017) 150–159
156
also play an important role in the availability and uptake of Fe from
soil, which is highly dependent on the nitrogen status of the soil
(Aciksoz et al., 2011; Oburger et al., 2014). Aciksozet al. (2011)
reported that increase in nitrogen supply could enhance the root release
of phytosiderophores in wheat plant. In the present study, increase in
the nitrogen fixing potential in treatments inoculated with sewage
grown microalgal consortia and enhanced siderophore production
could be a possible reason for the enhanced micronutrient (Fe) uptake
by the wheat plant. This aspect needs further investigation.The amount
of Fe and Zn mobilized to the grains was positively correlated (r =
0.89, 0.87) with the amount of N fixed in soil at mid-crop stage. The
iron concentration in plants ranges from 10–500 ug Fe /g dry weight
and graminaceous monocots are known to release phytosiderophores
for effective uptake of iron (Pii et al., 2016).
Cyanobacteria also have a role in transformation of soil micronu-
trients (Fe and Mn) available to the plants along with the production of
organic acids with chelating properties (Das et al., 1991). These
chelating compounds can increase the availability of micronutrients
to the growing plant. Several cyanobacteria viz. Anabaena flos-aquae, A.
cylindrica, Synechococcus leopoliensus, Anacystis nidulans are reported to
excrete strong copper-complexing agents (cK>108), while eukaryotic
algae only found to release weak copper-complexing agents
(cK<107.5). These copper complexing agents from microalgae may
control the speciation of copper in aquatic ecosystem (lakes) during or
after blooms (McKnight and Morel, 1980); however, their role in soil
micronutrient dynamics is far from understood. Therefore, in-depth
studies are required to understand the mechanisms involved in soil
micronutrient dynamics involving siderophore production, and cross-
talk between plant, microalgae and other microorganisms for micro-
algae/cyanobacteria based biofertilizer production.
4.2. Effect of sewage grown microalgal consortia on soil microbiological
parameters
In our study, T1 (Recommended dose of fertilizers) showed lowest
soil chlorophyll content, which reveals the negative impact of chemical
fertilizer on soil microbial photosynthetic activity, while that micro-
algae inoculation improved the chlorophyll content and nitrogen
fixation in soil cores. Soil organic carbon forms the basis of sustainable
agriculture and increase in organic carbon can improve soil health and
also help in the mitigation of atmospheric CO2 (Lal, 2004; Sá et al.,
2017). It is used as a source of energy for growth of plant and facilitates
the availability of nutrients through mineralization. Microalgal con-
sortia inoculated treatments recorded 35 – 51% higher soil organic
carbon as compared to control with recommended dose of fertilizer.
Dehydrogenases are one of the most important enzymes, occurring
in all living microbial cells, and therefore, used as an indicator of
overall soil microbial activity (León et al., 2017). They have a major
role in the biological oxidation of organic matter, representative of
microbial oxidative activities in the soil (Zhang et al., 2010). Soil
dehydrogenase activity is directly related to the physical, chemical and
biochemical properties of soil, hence a useful index of the soil fertility
and health (Salazar et al., 2011). In the present study, inoculation of
microalgal consortia showed an increase in the dehydrogenase activity
as compared to uninoculated treatments. The treatments- T4 (75% N +
full dose PK + MC1) exhibited highest dehydrogenase activity followed
by T5 (75% N + full dose PK + MC2). Higher dehydrogenase activity
in T4 (75% N + full dose PK + MC1) may be a net result of the higher
bacterial load in T4. A strong positive correlation was observed
between soil organic carbon and dehydrogenase activity (r = 0.77).
Salazar et al. (2011) also described the positive correlation of dehy-
drogenase activity with soil carbon, as observed in our study. However,
under anaerobic conditions in soil, high dehydrogenase activity can
impair Fe availability to the plants due to utilization of Fe (III) as
terminal electron acceptors in metabolic processes under waterlogged
conditions, by the soil microbe (Benckiser et al., 1984, Das and Varma,
2010).
4.3. Effect of sewage grown microalgal consortia on plant growth
parameters and yield
An increase in plant growth and yield was recorded in T4 and T5 as
compared to other treatments. Leaf chlorophyll was significantly higher
in T5 (75% N + full dose PK + MC2) with 53% increase in comparison
with the control. Chlorophyll content in leaf showed a strong positive
correlation with Fe (r = 0.92) and Cu content (r = 1.00) in shoot at
mid-crop stage. Leaf chlorophyll was also positively correlated with soil
Mn concentration (r = 99). The findings in the current study revealed
an increase in plant growth, yield and improvement in nutritional status
with the inoculation of sewage grown microalgal consortia. Similarly,
in previous studies, the inoculation of various cyanobacteria in wheat
plants increased plant chlorophyll to significant levels and improved
the nutritional status of plant (Prasanna et al., 2013). Recently,
Coppens et al. (2016) revealed that microalgae biomass obtained after
wastewater treatment can be used as a slow release fertilizer in tomato
crop. They also reported that microalgae inoculation improved the fruit
quality of tomato via increase in sugar and carotenoid content;
however, yields were poorer (Coppens et al., 2016). Garcia-Gonzalez
and Sommerfeld (2016) observed that the dry biomass and/or cellular
extracts of green alga Acutodesmusdimorphus also enhanced the germi-
nation rate, growth and floral production in tomato plant. Wuang et al.
(2016) illustrated the potential of Spirulina biomass grown in aqua-
culture wastewater for use as biofertilizer in leafy vegetables. They
found that application of Spirulina based biofertilizer enhanced the
plant growth and improved the seedling dry weight (Wuang et al.,
2016). Microalgae/cyanobacteria also produce plant growth promoting
substances (growth hormones), which improves the growth and overall
health of plant (Karthikeyan et al., 2007; Perez-Montano et al., 2014).
In the present study, crop yield was positively correlated with micro-
nutrient concentration of soil at mid-crop stage. A positive correlation
was found between spike number and Zn (r = 0.90) and Fe concentra-
tion (r = 91). Grain yield was also positively correlated with Zn (r =
0.80) and Fe (r = 87) content in soil at mid-crop stage. Therefore, it can
be envisaged that availability of Zn and Fe content in soil is crucial for
getting high yields in wheat crop. The effect of wastewater grown
microalgae biomass on soil and plant for biofertilizer application may
vary depending upon the quality of microalgae biomass and associated
bacteria; which is also highly dependent on the type of wastewater
used. Further studies on microalgae biomass generated using waste-
water from different sources and their field scale evaluation can help in
integrating them into nutrient management strategies for this crop.
5. Conclusions
The present study demonstrated the promise of sewage grown
microalgal consortia of filamentous and unicellular strains for enhanced
micronutrient availability in soil and micronutrient enrichment of
wheat crop. Significant increase in soil micronutrient content, particu-
larly Zn, Fe, Cu and Mn were recorded with consortia compared to
treatment with recommended dose of fertilizer, which can be related to
the positive interaction of plant and microalgae and stimulation of
siderophore activity in either plant/microalgae. However, this aspect
needs in-depth research, especially related to the release of metal
sequestering molecules by plants/microalgae. Both the microalgal
consortia, comprising either unicellular or filamentous native micro-
algae led to an increase in plant growth in terms of chlorophyll, besides
improving plant nutritional status, and enhancement in grain yield and
grain micronutrient content, illustrating their promise, particularly the
consortium of filamentous strains.The study highlighted that sewage
grown microalgae consortia can be suitable options for increasing crop
productivity, nutritional value and quality of wheat grains, besides
improving micronutrient availability in soil, through efficient mobiliza-
N. Renuka et al. Rhizosphere 3 (2017) 150–159
157
tion and translocation to the grains by the crop.
Acknowledgements
The first author is thankful to University Grants Commission, New
Delhi (UGC Reference No. F.151/2007(BSR) dated 23/03/2011) for
providing financial support to carry out the present experimentation.
All the authors are thankful to the Department of Botany, Panjab
University, Chandigarh; Division of Microbiology, Division of
Agronomy and National Phytotron Facility, ICAR-Indian Agricultural
Research Institute, New Delhi for providing the research facilities to
carry out the present investigation.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.rhisph.2017.04.005.
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	Wastewater grown microalgal biomass as inoculants for improving micronutrient availability in wheat
	Introduction
	Materials and methods
	Microalgal consortia used and their growth
	Experimental design
	Analyses of soil parameters
	Analyses of plant parameters
	Statistical analyses
	Results
	Micronutrient content in soil and plant samples
	Soil microbiological parameters
	Plant parameters
	Correlation between plant and soil micronutrient characteristics and yield parameters
	Discussion
	Effect of sewage grown microalgal consortia on micronutrient content in soil and plant samples
	Effect of sewage grown microalgal consortia on soil microbiological parameters
	Effect of sewage grown microalgal consortia on plant growth parameters and yield
	Conclusions
	Acknowledgements
	Supporting information
	References