Immobilization-Based Bio-formulation of Aspergillus awamori S29 and Evaluation of Its Shelf Life and Re-usability in the Soil–Plant Experiment

Abstract

The present study was an attempt to evaluate the bio-formulations of phosphate-solubilizing fungus Aspergillus awamori S29 using two economically viable carriers (calcium alginate and agar) in repeated batch fermentation. Further, the viable cell count under storage and response of these stored bio-formulations on the growth of wheat plants were studied at the end of 2, 4, and 6 months of incubation. Also, the response of these formulations in next season on pearl millet (bajra) was studied without further inoculation. In repeated batch fermentation assay, immobilized form performed significantly better than free form. The viability of fungal inoculant was 88.2% in calcium alginate-based bio-formulation after six months of storage. These bio-formulations showed not only a statistically significant increase in the growth of wheat crop in first season but also of pearl millet in next season. This work strengthens the re-usability potential of immobilized bio-formulations for next season crop.

Full text

Vol.:(0123456789)
1 3
Current Microbiology (2022) 79:163
https://doi.org/10.1007/s00284-022-02854-0
Immobilization‑Based Bio‑formulation ofAspergillus awamori S29
andEvaluation ofIts Shelf Life andRe‑usability intheSoil–Plant
Experiment
RachanaJain1,2· AnumeghaGupta2· VinaySharma2,3· SatyanarayanNaik1· JyotiSaxena2,4· VivekKumar1·
RamPrasad5
Received: 28 September 2021 / Accepted: 24 March 2022 / Published online: 18 April 2022
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2022
Abstract
The present study was an attempt to evaluate the bio-formulations of phosphate-solubilizing fungus Aspergillus awamori
S29 using two economically viable carriers (calcium alginate and agar) in repeated batch fermentation. Further, the viable
cell count under storage and response of these stored bio-formulations on the growth of wheat plants were studied at the
end of 2, 4, and 6months of incubation. Also, the response of these formulations in next season on pearl millet (bajra) was
studied without further inoculation. In repeated batch fermentation assay, immobilized form performed significantly better
than free form. The viability of fungal inoculant was 88.2% in calcium alginate-based bio-formulation after six months of
storage. These bio-formulations showed not only a statistically significant increase in the growth of wheat crop in first season
but also of pearl millet in next season. This work strengthens the re-usability potential of immobilized bio-formulations for
next season crop.
Introduction
For sustainable agriculture, the replacement of chemical fer-
tilizers with environment-friendly biofertilizers is the fore-
most requirement. Every biofertilizer has two components,
one is active and the other is inactive. The first active ingre-
dient is pure, viable microbial culture. The conservation of
these cultures in a metabolically and physiologically fit state
during storage and after application in the field is the main
challenge in the development of bio-formulations [1–3].
The second inactive component, called carrier, helps in the
enhancement of shelf life and efficiency of bioinoculant(s).
Presently, many forms of bio-formulation are available in
the market in liquid and solid states with their own pros and
cons. For instance, dry formulations have longer shelf lives
and are easy to store and transport, while liquid formulations
are faster to approach the plant system [1, 4–9].
In-depth research on phosphate-solubilizing fungi (PSF)
is currently ongoing worldwide, but the most PSF show
inconsistent performance in the field conditions [10–12].
The primary cause is the decline in the number of functional
cells during storage and secondly, the difficulty in coloni-
zation and survival in the field due to various biotic and
abiotic factors. The use of freshly prepared bioinoculant is
not always possible, especially when the bioinoculants are
commercialized. Therefore, there is a dire need to develop
a suitable carrier which can work as a protective niche to
increase the shelf life of fungi and also in fighting against
various biotic and abiotic factors during storage as well as
in field [13–17]. Bio-encapsulation as a carrier not only pro-
vides a protective environment during field and storage but
also assists the slow release of microbes.
* Jyoti Saxena
jyotisaxena2000@yahoo.co.in
* Ram Prasad
rpjnu2001@gmail.com
1 Centre forRural Development andTechnology,
Indian Institute ofTechnology, Delhi, Hauz-Khas,
NewDelhi110016, India
2 Department ofBioscience & Biotechnology, Banasthali
University, Tonk, Rajasthan304022, India
3 Amity Institute ofBiotechnology (AIB) Amity University,
Jaipur, Rajasthan303002, India
4 Biochemical Engineering Department, B.T, Kumaon Institute
ofTechnology, Dwarahat, Uttarakhand263653, India
5 Department ofBotany, Mahatma Gandhi Central University,
Motihari, Bihar845401, India

R.Jain et al.
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163 Page 2 of 10
There are also many reports where encapsulation were
used as bioinoculation strategy in soil–plant experiments
viz., calcium alginate (Ca alginate)-entrapped form of PSF,
Yarowia lipolytica and nitrogen-fixing bacteria Rhizobium
trifoli in Trifolium repens [18]; Ca alginate-encapsulated
and skim milk-amended inoculum preparation of PGPR,
Enterobacter sp. in Lactuca sativa [19]; agar-entrapped
Enterobacter sp. in onion [20]; Ca alginate beads of Bacil-
lus subtilis (NRRL B30408) and Pseudomonas corrugata
(NRRL B-30409) in wheat [21]; agar and calcium alginate-
immobilized form of Aspergillus awamori S19, A. tubingen-
sis S33, and A. niger S36 in mungbean [14, 22], and alginate
microcapsule of Klebsiella oxytoca Rs-5 in cotton [23]. Van
Elsas etal. [13] observed high-density colonization after
one week of encapsulated cell inoculation in wheat. Many
authors demonstrated that the encapsulated microbial forms
promoted plant growth similar or equivalent to their respec-
tive free cells [14, 22, 24, 25].
Thus, undoubtedly encapsulation of inoculants can
improve effectiveness, but hardly any reports are avail-
able where further shelf life and effectiveness of fungal
bio-formulations after storage were studied. However, such
studies can be of great help in the commercialization of the
entrapped form-based bio-formulations. Keeping the above
in mind, the present investigation focused on following: a)
the preparation of carrier-based bio-formulation of Aspergil-
lus awamori S29 using Ca alginate and agar as carrier mate-
rials; b) checking of their potential in flask culture method;
c) further, testing the shelf life of bio-formulations at 2,
4, and 6months and their efficacy on growth and yield of
Triticum aestivum (wheat) plants in a pot experiment; and d)
finally, the efficacy of the biofertilizer/bio-formulation was
studied in next season by growing Pennisetum glucum (pearl
millet; bajra) plants in same soil without adding further bio-
formulations. The selection of wheat as the first crop and
pearl millet as a second crop in the pot experiments was
made due to seasonal constraints of Rabi and Kharif crops.
The first experiment was executed in winter season with
wheat and in the subsequent season, pearl millet was used.
Materials andMethods
Fungal Strain
The microorganism, Aspergillus awamori S29 used in this
study was earlier isolated from rhizosphere soil of Vigna
radiata grown in the semi-arid environment of Banasthali,
Rajasthan. It was identified by colony morphology and phe-
notypic characteristics and further by molecular method
using ITS1-5.8S-ITS2 region sequencing and phylogenetic
analysis. The culture was submitted to IMTECH Chandi-
garh (accession number MTCC 9630). The fungal strain was
previously studied for its phosphate solubilization potential
under alkaline semi-arid conditions [26] in broth as well as
in the soil–plant system and showed great potential to be
used as biofertilizer. This paper focuses on the development
of a better bioinoculation strategy for S29 strain by immo-
bilization with higher shelf life and long-term effectivity in
the soil–plant system.
Immobilization ofFungal Strain
The fungal strain was cultured on Potato Dextrose Agar
(PDA) slant for five days and then used for the preparation
of three bio-formulations: (1) Ca alginate beads, (2) agar
blocks, and (3) broth (free form), following the method
described in Jain etal. [14]. Firstly, spores were suspended
in Tween-800 (0.1%) to prepare a final solution with spore
concentration of 2 × 109 spores ml−1. Three ml of this spore
suspension was mixed in 100ml of 3% sodium alginate
solution. The mixture obtained was extruded drop-wise into
0.5M calcium chloride solution with the help of a syringe
(syringe size 25ml; needle size 0.56mm ~ 22mm).
For agar entrapment, 3ml of spore suspension was mixed
in 100ml of 3% solution of agar (by melting 3g agar in
distilled water and making the volume up to 100ml). It was
then poured in a sterile Petri dish to form a thin layer of
about 2mm. After solidification the medium was cut with
sterile sharp-edged knife into 3mm × 2mm pieces. The
pieces were hardened in olive oil. Both beads and blocks
were thoroughly washed 5–6 times with sterile distilled
water and used for further studies. One ml beads/blocks were
transferred in 0.2M phosphate-buffered saline (PBS) of pH
7.0 for cell counts to check the entrapment efficiency. The
beads/blocks were placed in sterile flask, sealed, and stored
for further use.
Batch Fermentation
For batch fermentation, two media were prepared; one was
growth medium in which the microbes were grown and
another was the production medium in which insoluble P
(Tri-calcium phosphate, TCP) was added to check the phos-
phate solubilization. Repeated batch fermentation was per-
formed for five cycles using TCP as an insoluble P source, as
reported by Jain etal. [14]. Approximately 120ml beads of
2–3-mm size were prepared by extrusion of 100ml sodium
alginate solution in calcium chloride. Six ml gel spore beads/
blocks (1ml beads contain around 50–55 beads and 1ml
agar blocks contain around 30–32 blocks) were submerged
in 60ml growth medium in 250ml Erlenmeyer flasks and
incubated at 30 ± 2°C in an orbital incubator shaker for 24h
at 130rpm. Free mycelium cultivation was carried out in
flasks with 60ml growth medium inoculated with 146µl
of A. awamori spore suspension having 5 × 109cfu ml−1

Immobilization‑Based Bio‑formulation ofAspergillus awamori S29 andEvaluation ofIts Shelf…
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concentration. The gel beads were then separated from the
growth medium after 24h by filtrations, washed with sterile
distilled water, and transferred into 60ml fresh production
medium in flasks. Production medium was changed follow-
ing the same procedure every 48h and the filtrate was used
for further analysis. Soluble P was analyzed by the molyb-
denum blue method [27]. Glass electrode pH meter was used
for pH measurements. The titratable acidity (TA) was meas-
ured by titration [28].
Shelf‑Life Assessment
The shelf life of biofertilizer is one of the primary criteria in
biofertilizer development researches. The Ca alginate beads,
agar blocks, and the free form broth were stored in the her-
metically sealed sterile flasks and stored at 4°C at room
temperature for 6months. The whole preparation (immo-
bilization in the Ca alginate and agar) was carried out at
regular time intervals of 2, 4, and 6months. The number
of viable spores was determined using a standard dilution
plate count method. For this, samples were collected from
different stored bio-formulations at 2-month intervals for
six months under completely sterilized conditions. One ml
beads/blocks were suspended in 9ml of 0.2M phosphate-
buffered saline (pH 7.0) and incubated at room temperature
for 30min. Further, it was crushed in a mortar and pes-
tle by light hand to release the entrapped spores into the
solution. It was serially diluted and plated on Pikovskaya
(PVK) agar plates and incubated at 28°C for four days. The
viable spores were counted using the colony counter. All the
experiments were performed in triplicate.
Soil–Plant Experiment
Efficacy Evaluation oftheBio‑formulation Under
Greenhouse Conditions withTriticum aestivum
(Wheat)
The experiment was performed in plastic pots with unsterile
field soil (clay 0–25mm; pH 8.1; electric conductivity 0.28
dS m−1; organic matter 0.78%; available P 5.08kg ha−1; K
280kg ha−1) in a greenhouse without any temperature or
illumination controls and subjected to environmental condi-
tions. Two types of pot mix were prepared viz., soil with-
out amendment and soil with TCP amendment. There were
20 treatments (three replicates of each) viz., (1) Soil; (2)
Soil + F (Free form of Fungi); (3) Soil + B (Calcium algi-
nate-entrapped form of A. awamori) Fresh; (4) Soil + B (2
Months); (5) Soil + B (4 Months); (6) Soil + (6 Months); (7)
Soil + A (Agar entrapped form of A. awamori in block form)
Fresh; (8) Soil + A (2 Months); (9) Soil + A (4 Months); (10)
Soil + A (6 Months); (11) Soil + TCP; (12) Soil + TCP + F;
(13) Soil + TCP + B (Fresh); (14) Soil + TCP + B (2 Months);
(15) Soil + TCP + B (4 Months); (16) Soil + TCP + B (6
Months); (17) Soil + TCP + A (Fresh); (18) Soil + TCP + A
(2 Months); (19) Soil + TCP + A (4 Months); and (20)
Soil + TCP + A (6 Months).
Wheat (Triticum aestivum cv. RAJ 3765) seeds were
obtained from Krishi Vigyan Kendra, Banasthali University
and surface sterilized by sodium hypochlorite solution (1%)
for 20min and rinsed with sterile water. Bio-formulation
application was done as mentioned in Jain etal. [14]. The
plant was uprooted after 90days of sowing and measure-
ments on length and dry weight of root and shoot and spike
weight were recorded.
The Efficiency ofBio‑formulation inNext Season
withPennisetum glaucum (Pearl Millet)
In next season, Kharif crop pearl millet was grown in the
same pot having same soil, without further addition of bio-
formulations. Seeds were surface sterilized in a sodium
hypochlorite solution (1%) for 20min and rinsed with ster-
ile water. Pearl millet plants were harvested after 50days
of sowing and their shoot and root lengths and dry weights
were recorded.
Statistical Analysis
The experiments were performed in a completely rand-
omized design. Mean and Standard Deviation were calcu-
lated for each parameter in each treatment. Further, means
were analyzed by ANOVA and compared by Duncan’s mul-
tiple range test (p < 0.05).
Results
Efficacy oftheImmobilized Formulation oftheA.
awamori S29 inRepeated Batch Fermentation
Table1 clearly depicts that the immobilized fungi were sig-
nificantly (p < 0.05) more effective TCP solubilizer than free
cells. The average amount of soluble P in the broth was 351
and 347mg l−1 batch−1 cycle−1 attained by Ca alginate and
agar-encapsulated S29, respectively. The free form showed
significantly (p < 0.05) lower pH and high TA in comparison
to the immobilized form, but the phosphate solubilization
was significantly (p < 0.05) higher in immobilized forms.
In these two immobilized forms, the maximum soluble P
levels of 408 (Ca alginate entrapped) and 391mg l−1 (agar-
entrapped form) were achieved in fifth cycle (Fig.1).

R.Jain et al.
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163 Page 4 of 10
Entrapment Efficiency andShelf Life
oftheBioinoculants During Storage
oftheFormulations
Entrapment efficiency represents the actual percentage of
entrapped spores over the initial spore percentage used
to prepare the formulation. In case of Ca alginate, 98.2%
entrapment efficiency was recorded, whereas in agar
immobilization, it was 99.7%. Further, the shelf life of
free and entrapment-based bio-formulations was studied
at two different temperatures viz., low temperature (4°C)
and room temperature (28°C) for six months using dilu-
tion plate count method. Results are given in Fig.2 which
clearly show the effect of immobilization on the viable cell
number. The survival rate of the low-temperature-stored
immobilized bio-formulation remained constant even in
free form and non-significant decline in spore viability
was observed throughout the storage period. On the other
hand, at room temperature immobilized bio-formulation
showed non-significant decline in cell viability, while sig-
nificant decline was observed in free form. The shelf life of
the immobilized formulation was significantly higher than
the free form. It was noted that Ca alginate formulation
had better viability than the agar-based bio-formulation.
At room temperature, Ca alginate beads showed a mini-
mal decline in the viability of the spores; the initial spore
count of freshly prepared bio-formulation was found to be
4.91 × 109 which decreased to 4.33 × 109 after six months
of storage. This was followed by agar blocks in which
the initial viable spore count was 4.95 × 109 and after six
months it was reduced to 3.92 × 109. The major decrease
was noticed in free form where after six months the viabil-
ity count was obtained as 2.82 × 109 (initial being 5 × 109).
Thus, Ca alginate, agar entrapped, and free form showed
88.2%, 79.3%, and 56.4% viable spores, respectively, after
Table 1 Average value of pH, soluble P (mg l−1), and titratable acid-
ity (mmol H+ l−1) with free and immobilized forms of A. awamori
S29 on production medium with TCP after 5 repeated cycles
a Each value is the mean of three replicates followed by a standard
error. Mean values within a column followed by the same letter are
not significantly different at p ≤ 0.05
b A29—Agar-immobilized A. awamori S29; B29—Ca alginate-immo-
bilized A. awamori S29; F29—Free form of A. awamori S29
Fungal Formb) pHaSoluble P
(mg l−1cycle−1)
Titrable acid-
ity (mM H+
l−1)
A29 4.57 ± 0.02b347 ± 06.79a28 ± 1.69b
B29 4.53 ± 0.06b351 ± 04.78a30 ± 0.95ab
F29 4.40 ± 0.12a300 ± 17.68b33 ± 5.53a
Fig. 1 Solubilization of tri-calcium phosphate by Aspergillus awamori
S29 encapsulated in agar, calcium alginate, and free cells (Control)
during repeated batch. Data are means of three replicates. Mean values
with the same letters do not differ significantly by Duncan’s multiple
range test at p ≤ 0.05
Fig. 2 Shelf-life study of Ca
alginate, agar entrapped, and
free form of A. awamori S29
bio-formulations stored at 4 and
28°C for six months. Data are
means of three replicates. Mean
values with the same letters
do not differ significantly by
Duncan’s multiple range test
atp ≤ 0.05

Immobilization‑Based Bio‑formulation ofAspergillus awamori S29 andEvaluation ofIts Shelf…
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Page 5 of 10 163
six months of storage at room temperature. Consequently,
Ca alginate beads were found to be the most robust among
the three bio-formulations in terms of sustaining the maxi-
mum number of spores for the longest duration of time.
Evaluation ofEfficacy oftheBio‑formulations Under
Soil–Plant Condition
Efficacy oftheBio‑formulations onTriticum aestivum
The growth of T. aestivum was measured by recording the
lengths and dry weights of root and shoot and spike weight
after 90days from germination (Fig.3 a–e). The pots with-
out any inocula were used as control 1, and pots with freshly
prepared free form inoculants were used as control 2.
Phosphate-solubilizing fungi, A. awamori showed sig-
nificant effect on growth parameters of wheat in comparison
to uninoculated control 1. In both pot mixes, each treated
pot showed a significant (p < 0.05) increase in shoot length
and dry weight in contrast to control 1. When the wheat
growth was analyzed for immobilized and free form inocu-
lants (control 2), the entrapped form-treated plants had
either significantly (p < 0.05) higher or equivalent outcome.
With TCP, four-month-stored Ca alginate-entrapped treat-
ment showed highest increase in shoot length (39%), while
two-month-stored form showed maximum rise in shoot dry
weight (191%) over the control 1.
In all pots, a non-significant difference in root dry
weight was observed. In pots containing only soil, the agar-
entrapped fungus showed a significant (p < 0.05) increase in
root length over the uninoculated control 1 and free form-
treated control 2. On the contrary, the highest root length
with 69% increase was noted in TCP-enriched soil with the
freshly prepared Ca alginate-immobilized treatment.
The plants grown in pots having only soil showed that
all fungal-treated plants presented significantly (p < 0.05)
higher spike weight in contrast to control 1. In compari-
son to control 2, agar-entrapped fungi showed significantly
(p < 0.05) higher spike weight. The highest rise in spike
weight was observed in the 2-month-old agar-entrapped
form. It showed 139 and 51% increase in comparison to
control 1 and control 2, respectively. With TCP, all treated
plants showed significantly higher spike weight in contrast
to uninoculated control. Ca alginate 4-month-stored form
presented significantly (p < 0.05) highest spike weight and
was followed by agar-entrapped form. Ca alginate-entrapped
6-month-stored form showed 286% and 65% higher spike
weight in comparison to control 1 and control 2, respec-
tively. Overall, immobilized form was found significantly
better or equivalent compared to the free form of the fungal
isolate.
Re‑usability Efficacy oftheCarrier‑Based
Formulations oftheA. awamori S29 intheSoil–Plant
Experiment
Most of the work on bio-formulation preparation available
in literature has been focused on the survival of inoculum
during storage and often ignored to test the effectiveness
of inocula for the desired activity in the next season. In the
present investigation, the effectiveness of bio-formulation
was checked in the next season without further inoculating
the bio-formulation in the pots to understand the re-usa-
bility of the inocula. Therefore, the same pots of previous
experiment were used for growing the next season crop.
For this experiment, the Kharif crop, pearl millet was
selected due to seasonal constraints. It is one of the major
crops of Banasthali region. Surprisingly, both the forms,
fresh and stored, performed well in the next season (Sup-
plementary material Figs. S 1 and 2). Figure 4 (a–d)
depicts that, in general, the plant growth was significantly
(p < 0.05) enhanced by the immobilized forms of fungi in
comparison to the free form (Control 2) and uninoculated
control 1 in the next season.
In both the unamended and TCP-amended soil, stored
as well as freshly prepared Ca alginate and agar-entrapped
forms demonstrated significant rise in shoot length
(7–56%) and shoot dry weight (10–203%) in contrast to
control 1 plants. In soil without amendment, 6-month-
stored Ca alginate-entrapped form showed significantly
(p < 0.05) higher shoot length with 51% and 42% increase
in comparison to control 1 and control 2, respectively.
However, in case of TCP-amended soil, 4-month-stored
Ca alginate-entrapped form showed significantly (p < 0.05)
higher shoot length as compared to control 1 and control
2. As regarding shoot dry weight, the 4- and 6-month-
stored Ca alginate and agar-entrapped forms showed a sig-
nificantly (p < 0.05) better rise. In TCP-amended soil, the
agar-stored form performed significantly (p < 0.05) better
than others in terms of shoot dry weight. These results
indicated that the stored forms performed significantly
(p < 0.05) better than the freshly prepared immobilized
forms.
In pots holding only soil, mainly all agar-entrapped
forms of S29 (stored and fresh) showed a significantly
(p < 0.05) higher root length in contrast to Ca alginate-
based treatment (except in 4-month-stored form) and con-
trol 1 & 2. Similarly, significantly higher root dry weight
was shown by all forms in comparison to control. With the
TCP, although the root dry weight was significantly similar
in all treated as well as untreated plants but root length
significantly (p < 0.05) increased in all the immobilization-
based bio-formulations of S29 (except 2months stored and
freshly prepared forms of agar).

R.Jain et al.
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163 Page 6 of 10
Fig. 3 Effect on Triticum aestivum plant growth and yield after inocu-
lating with stored Ca alginate, agar immobilized (2, 4, and 6months),
freshly prepared immobilized, and free form formulation of A.
awamori S29 in TCP amended and unamended soil. (a) Shoot length,
(b) shoot dry weight, (c) root length, (d) root dry weight, and (e) pod
weight. Data are means of five replicates. Mean values with the same
letters do not differ significantly by Duncan’s multiple range test at
P ≤ 0.05

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Principle Component Analysis
The principle component analysis was performed to assess
the statistical correlation between different treatments and
parameters (Fig.5). For different treatments, plant growth
and yield are explained with component 1 (PC1: 73.28%)
and component 2 (PC2: 13.53%). Results from this study
suggested that application of bioinoculants significantly
affected the plant growth and yield in plants. Significant
contribution to PC1 was due to root and shoot dry weight,
yield, and shoot length with values of 0.484, 0.477, 0.484,
and 0.447, respectively. However, root length had the
maximum contribution to PC2, with a value of 0.853. The
results also revealed that application of organic manures
alone was less effective than combined effect of organic
manures and biofertilizers. Among the different treatments
TCP + Unimmobilized, TCP + B29 Fresh, TCP + B29 + 2M,
TCP + B29 + 4M, TCP + B29 + 6 M, TCP + A29 + 2 M,
TCP + A29 + 4 M, TCP + A29 + 6 M, and S + Unimmo-
bilized showed positive effect on plant growth and yield
parameters. The PCA analysis showed that all studied attrib-
utes were affected by treatments but major effect was on
shoot dry weight, root length, and yield.
Discussion
In the present study, TCP was used as a source of insoluble
P because the experimental site, Banasthali is a semi-arid
region of Rajasthan and the soil is calcareous and Phos-
phorus is majorly present in calcium-bound insoluble form.
Fig. 4 Effect on bajra growth, after growing in the same pot of stored
immobilized (2, 4, and 6months), freshly prepared immobilized, and
free form formulation of A. awamori S29 without further inocula-
tion and amendment. (f) Shoot length, (g) shoot dry weight, (h) root
length, and (i) root dry weight in next season. Data are means of five
replicates. Mean values with the same letters do not differ signifi-
cantly by Duncan’s multiple range test at p ≤ 0.05

R.Jain et al.
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163 Page 8 of 10
Compared to free form, immobilized cultures of A.
awamori S29 showed higher levels of TCP solubilization
in fermentation medium. These results are consistent with
those of El-Komy [29], Jain etal. [14, 22], and Minaxi and
Saxena [25]. All these studies reported improved phosphate
solubilization by Ca alginate and/or agar-immobilized
Azospirillum lipoferum, A. awamori, Bacillus megaterium,
Pseudomonas fluorescens, and Burkholderia cepacia in
comparison to free cell suspension. The results presented
here clearly illustrated that immobilization of inocula could
significantly enhance the P solubilization efficiency and re-
usability in comparison of freely suspended cells.
Further, the entrapped efficiency (%) of A. awamori S29
in Ca alginate and agar was also studied during the investiga-
tion by determining the difference between the number of
cells added in the initial solution and successfully entrapped
cells in final gel beads/blocks after the hardening process. In
case of Ca alginate, 98.2% entrapment efficiency was found
which is comparable to Gul and Dervusoglu [30] report for
Ca alginate-entrapped Lactobacillus casei shirota. In agar
immobilization, 99.7% entrapment efficiency was achieved
which is almost similar to the results of Lotfipour etal. [31]
for Ca alginate immobilization of L. acidophilus (99.8%) and
Bassani etal. [32] for E. coli and E. aerogenes.
The shelf life of the bio-formulation during storage is a
crucial factor in the commercialization of bio-formulation.
Encouraging results were obtained in shelf-life study at
4°C as well as room temperature after two, four, and six
months of storage. Here, the number of viable spores in the
Ca alginate beads and agar block remained constant at 4°C,
while a decline in viable number ofspores was observed in
case of free form. On the other hand, at room temperature
both immobilized as well as free bio-formulations showed
decline in viable cell count but the carrier-based formula-
tion had higher viable spores than free form. Duan etal.
[33] also reported similar observations that alginate beads
of Paecilomyces lilacinus and P. chlamydosporia showed
substantially more stable viability when stored at low tem-
peratures (4°C) than at room temperature. Our results are
better than Kaushik etal. [34] who observed only 68%
survival in MG1 myco-granules of Aspergillus lentulus at
room temperature. The reason behind higher shelf life of
immobilized form is the protection of the cell inside the
bead from the harsh external environment, contamination,
and pathogens [35–37]. In other words it can be said that
immobilization successfully generated a low-stress micro-
environment. These results are very favorable with respect
to the development of a novel biofertilizer product [38–40].
Nevertheless, greenhouse experiments are neces-
sary to evaluate the performance of the bio-formulation
in a soil–plant system. In the soil–plant experiments, an
increase in wheat growth was observed by inoculation with
A. awamori S29 in first season as compared to uninoculated
control and the performance of immobilized form was better
than the free form. These results are in corroboration with
various earlier reports of Vassilev etal. [41]; Rekha etal.
[24]; Jain etal. [14, 22]; Minaxi and Saxena [25]; Guimarães
etal. [42]; and Klaic etal. [40].
It was also observed that stored immobilized form per-
formed either equivalent or significantly better than their
respective freshly prepared immobilized form. In contrast,
in the shelf-life study, we found a decrease in viable cell
count with time, but the impact of that was not visible in the
soil–plant experiment. A similar observation was made by
Fig. 5 Principal component
analysis showing correlation
between different treatments
and variables (plant growth and
yield attributes) of both seasons

Immobilization‑Based Bio‑formulation ofAspergillus awamori S29 andEvaluation ofIts Shelf…
1 3
Page 9 of 10 163
Bernabeu etal. [43] in the liquid formulation of Parabur-
kholderia tropica that a decline in the viability of bacteria
with respect to time did not actually affect its colonization
ability. The possible reason could be the protective niche that
was provided by the carrier and slow release of the fungal
cell helped the cells to colonize, and work efficiently even
when the number of viable cells was low. It was also enu-
merated that a six-month storage form was as good in plant
growth stimulation as freshly prepared form. Similar results
were reported for pesta formulations of Colletotrichum trun-
catum and Fusarium oxysporum revival and myco-herbicide
activity after 18months of storage at 4°C and five years of
storage at cold storage, respectively [44, 45].
In next season, immobilized form of inocula showed sig-
nificant enhancement in growth of pearl millet plant without
further addition of bio-formulation in the pots that proved
the re-usability potential of the immobilization-based bio-
formulations. It could be attributed to the protection pro-
vided against the biotic and abiotic stresses which helped
to retain sufficient number of viable cells up to next season.
To the best of our knowledge, this is the first report where
the impact of bio-formulations was studied in next season
without further adding the inoculum.
In the present work, the storage time was studied up to six
months, and the results showed promising viability not only
during storage but also in soil, although there are reports of
viability of fungal and bacterial inocula from 1 to 5years
[43, 44, 46]. The experiments related to study the shelf life
for longer periods are in offing.
Further, it can be concluded that alginate and agar can be
used to produce consistently performing fungal inoculants,
in terms of the beneficial effect of its formulation on plant
growth. This study will prove useful for future studies to
acquire more insight into bio-formulation production using
immobilization as a carrier development technology.
Conclusion
The A. awamori S29 formulations were successfully immo-
bilized in agar and Ca alginate as a carrier and used for
their efficacy in stimulating the growth of wheat and pearl
millet in the pot experiments for two consecutive seasons.
The results obtained in the present study not only showed
growth promotion in the season of application but also in
next season which in turn would save labor and energy and
also be economical.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00284- 022- 02854-0.
Acknowledgements Authors are grateful to the Vice-Chancellor of the
Banasthali University for providing the facilities to carry out the work.
Author Contribution RJ was PI of the project and performedlab work,
writing, and statistical analysis; AG did all the experimental work; VS
contributed to conceptualization; S.N. Naik contributed to reviewing
and editing of the manuscript; JS contributed to writing and editing of
the manuscript, RP contributed to review and editing of manuscript;
VK contributed to revision and editing of manuscript.
Funding This work was funded by the University Grant Commission
(UGC), India and Department of Science and Technology (DST) India.
Declarations
Conflict of interest The authors declare no conflict of interest.
Research Involving Human and Animal Participants This article does
not contain any studies with humans or animal.
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