3 Biotech (2020) 10:297

https://doi.org/10.1007/s13205-020-02281-7

ORIGINAL ARTICLE

Influence of biosurfactant producing Bacillus tequilensis LK5.4 isolate

of kinema, a fermented soybean, on seed germination and growth
of maize (Zea mays L.)
Lalit K. Chaurasia1 · Buddhiman Tamang1 · Ranjan K. Tirwa1 · Pinkey L. Lepcha1

Received: 4 September 2019 / Accepted: 29 May 2020 / Published online: 10 June 2020
© King Abdulaziz City for Science and Technology 2020

Abstract
In this study, the lipopeptide biosurfactant was extracted, purified and characterized from the Bacillus isolate LK5.4 obtained
from kinema samples of Sikkim. Plant growth-promoting property of the biosurfactant producing bacterium was also evalu-
ated. Out of fifty-seven isolates, only ten were biosurfactant producer as determined by the oil displacement test. Bacillus
isolate LK5.4 showed the maximum emulsification index (52.3 ± 0.02), reduced surface tension up to 40% and produced
754 mgL−1 biosurfactant in the nutrient broth. Based on 16S rRNA gene sequencing, the isolate LK5.4 was identified as B.
tequilensis. Biosurfactant was purified by Thin Layer Chromatography (TLC). Evaluation of the chemical characteristics by
TLC, Liquid Chromatography-Mass Spectrometry, Fourier Transform Infrared Spectroscopy and Nuclear Magnetic Reso-
nance Spectroscopy identified the biosurfactant as surfactin. The effect of different concentration of biosurfactant in maize
seed germination was evaluated under in vitro condition. It showed the fastest growth of seedlings at 300 µg/ml biosurfactant
solution. Similar results were shown by the potted plant experiment, where the soil was directly treated with biosurfactant
producing bacterium LK5.4. The LK5.4 treated plants showed a mean height of 29.17 ± 0.47 cm and mean leaf length of
18.42 ± 0.17 cm while the mean height and mean length of the leaf were 15.48 ± 0.98 cm and 11.12 ± 0.40 cm respectively
in the control plants. The treated plants had higher moisture content (68.48 ± 2.79%) than the control plants (50.53 ± 1.63%),
which is because of higher bioadsorption in the treated plants. These results provided indirect evidence of plant growth-
promoting property of the biosurfactant.

Keywords Bacillus tequilensis · Kinema · Biosurfactant · NMR · LCMS · Maize plant

Abbreviations Introduction
BS Biosurfactant
EI24 Emulsification index Surfactants have the unique characteristic to emulsify the
TLC Thin layer chromatography hydrocarbons in water (Desai and Banat 1997). Being
ESI & APCI MS Electrospray ionization and atmos- amphipathic, surfactants dissolved in the mixture of polar
pheric pressure chemical ionization and non-polar solvents accumulates at the interphase of the
mass spectrometry two solvents when kept undisturbed for a long time (Steinbu-
FTIR Fourier transform infrared chel 2011). This explains the emulsification, solubilization,
spectroscopy lubrication, phase dispersion and detergency properties of
NMR Nuclear magnetic resonance the surfactants (Deleu and Paquot 2004; Gautam and Tyagi
2006; Nitschke and Costa 2007). Though the chemically
synthesized surfactants are available in the market, the bio-
Electronic supplementary material The online version of this
surfactants (BS) are becoming more popular because of their
article (https​://doi.org/10.1007/s1320​5-020-02281​-7) contains
supplementary material, which is available to authorized users. low toxicity, higher biodegradability and environmental
compatibility (Cameotra and Makkar 1998). Among other
* Buddhiman Tamang sources, microorganisms can also produce a huge diversity
bmtamang3@gmail.com of BS either as membrane components or as secondary
1
Department of Microbiology, Sikkim University, 6th Mile, metabolites (Cao et al. 2009).
Samdur, Tadong, Sikkim, India

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BS has a great application in crop improvement and
agriculture (Bee et al. 2019; Sachdev and Cameotra 2013).
Recently, microbial BS are increasingly researched for enhanc-
ing the agriculture yield as they have been found to control
plant pathogens and increase the bioavailability of nutrients
for the plants (Kim et al. 2015). BS producer microorganisms
with the ability to degrade hydrocarbon pollutant have also
been found to promote the growth of the plants (Burd et al.
2000; Germaine et al. 2006; Kruijt et al. 2009; Sheng et al.
2008; Singh and Cameotra 2013). BS plays a significant role
in signalling, plant microbial interaction and biofilm formation
(Bee et al. 2019; Sachdev and Cameotra 2013). It also assists
in the inhibition of plant pathogens, enhancement of seed ger-
mination and also promotes the bioremediation of agricultural
soil (Sachdev and Cameotra 2013). Sheng et al (2008) showed
that Bacillus sp. J119 isolated from cadmium (Cd) contami-
nated soil can enhance the plant growth and also promotes the
colonization of plant growth-promoting rhizobacteria (PGPR)
(Sheng et al. 2008). Bacillus sp is already well known for its
direct antagonistic activity against many plant pathogens.
Besides, surfactin and fengycin produced by Bacillus sp stimu-
late the induced systemic resistance (ISR) phenomena against
plant pathogen and provide significant resistant to the plant to
protect them from pathogens (Sheng et al. 2008).
Kinema, the ethnic fermented soybean product of Sikkim,
Darjeeling hills (India), Nepal and Bhutan, is fermented by a
diverse group of Bacillus sp. (Sarkar et al. 2002) in addition
to Enterococcus faecium, Candida and Geotrichum (Sarkar
et al. 1994). During fermentation of kinema, the unsaturated
fatty acids (oleic, linoleic, and linolenic) increases by almost
six times (Sarkar et al. 1994). Because of high oil content,
Bacillus sp. of kinema has the capability to produce BS.
Generally the Bacillus isolates from the fermented foods
belong to GRAS (Generally regarded as safe) status and thus
the application of these bacteria is assumed safe and benefi-
cial, especially in Sikkim, which is an organic state. Focus-
ing on the above-mentioned properties, Bacillus sp. from
kinema were studied for production and characterization of
BS and its effect on corn germination and growth promotion
effect of BS producing bacterium on corn plants have been
studied. This study provides evidence for understanding the
role of BS in plant growth promotion and crop improve-
ment. This work also shows the potential of BS producing B.
tequilensis for the development of biofertilizers to augment
organic farming in this region.
Material and methods
Sampling and sample preparation
Kinema is an ethnic fermented food of Sikkim and Darjeel-
ing region of north-east India. It is prepared by fermentation
13
3 Biotech (2020) 10:297
of boiled soybean seeds with Bacillus sps. It is semisolid
with sticky texture because of polyglutamic acid produc-
tion (Chettri et al. 2016). Hydrolysis of soy proteins will
release ammonia which not only increases the pH (~ 7.9)
but also imparts mild ammonical flavour in the finished
product (Sarkar et al. 1994). Kinema is sold fresh in the
market wrapped in fig leaves (Ficus plant) or banana leaves
(Tamang 2015). It can be consumed fresh or dried in the sun
to increase the shelf-life. Thus, dried kinema can be stored
in the room temperature in an air-tight container for several
months (Tamang 2015).
Eight kinema samples were collected from the local mar-
ket of Gangtok, Sikkim for isolation of Bacillus. After pur-
chasing, the samples were immediately transferred to the
laboratory and stored in the refrigerator. Ten grams of kin-
ema samples were suspended in 90 ml of sterilized normal
saline and mashed in stomacher blender. The suspension
was heated in a water bath at 80 °C for 20 min to remove
other contaminants. As a result, only Bacillus spores would
survive in the sample.
Isolation and screening of BS and strain selection
The suspension was serially diluted till the concentration of
­10–4 and inoculated on nutrient agar plates by spread plate
method with the help of glass spreader. The plates were
incubated at 37 °C for 24 h. As a result, discrete colonies
developed on the plates. Pure cultures were obtained by the
streak plate method. The pure cultures were preserved in
30% glycerol solution at − 80 °C.
All the pure isolates were cultured in 250 ml nutrient
broth, under the above-mentioned conditions. After 48 h
incubation, the broth was centrifuged at 12,000 rpm for
20 min to separate the bacterial cells. The cell-free superna-
tant was used for the screening of isolates for BS production.
Oil displacement test was conducted according to Youssef
et al. with some modification (Youssef et al. 2004) and the
diameter of oil displacement was measured. To determine
the emulsification index, an equal amount of sunflower oil
and the cell-free supernatant was vortexed. The tube was
kept undisturbed for 14 h. The emulsification zone was
measured (Pereira et al. 2013). The surface tension was
measured by Du Noüy Ring Tensiometer (Kruss GmbH
Hamburg, Germany) (Jazeh et al. 2012). On the basis of
the above three screening methods, the isolate LK5.4 was
selected for identification.
Microscopic analysis, biochemical and sugar fermen-
tation tests were done for identification of bacteria as per
“Bergey’s manual of systematic bacteriology” (Vos et al.
2009). Genomic DNA was extracted by phenol-chloroform
method with some modification (Sambrook et al. 2006). 16S
rRNA gene was amplified with 27F and 1492R primers (Bee
et al. 2019; Sachdev and Cameotra 2013). 1 µl of purified
3 Biotech (2020) 10:297
PCR products were subjected to sequencing in automated
DNA sequencer (3500 Genetic Analyzer, Applied Biosys-
tems, Japan). The forward and reverse sequences obtained
from sequencing were assembled by Codon Code Aligner
software. The species was identified by 16S rRNA gene
sequencing (Bento et al. 2005). Nucleotide sequence identi-
ties were determined using the BLAST tool from the NCBI
nr/nt database. Partial sequence data for the 16S rRNA genes
have been deposited in the GenBank nucleotide sequence
data libraries (Bento et al. 2005) and their accession num-
bers were obtained.
Studies on the BS
After the identification, the Bacillus isolate LK5.4 was inoc-
ulated in one-litre nutrient broth for batch culture. The pH
of the media was maintained at 7 and incubation was done
at 37 °C for 48 h.
Extraction and purification of BS
The cells from the 48 h batch culture were removed by cen-
trifugation at 8000 rpm for 20 min. The supernatant was
acidified with 5 M HCl till the pH of the media reached the
value of 2.0. It was kept at 4 °C overnight. This allowed the
BS to precipitate and settled down at the bottom of the flask
which was harvested by centrifugation at 12,000 rpm for
20 min. Subsequently, the precipitate was suspended in chlo-
roform and methanol solution in 2:1 ratio and centrifuged
again. Crude BS was collected from the interphase of chlo-
roform and methanol (Varadavenkatesan and Murty 2013).
Crude BS sample was dissolved in methanol at the con-
centration of 100 µg/ml. About 10 µl of BS was spotted on
a silica gel TLC plate (Silica gel ­60F254, Merck, Germany).
The mobile phase was prepared with chloroform: methanol:
28% ammonia water in the proportion of 65:35:5. After the
development of chromatogram, the plate was treated with
0.5% ninhydrin in acetone and heated at 85 °C to develop
the spots (Varadavenkatesan and Murty 2013). Rf values of
BS bands were calculated (Sukirtha and Usharani 2013).
Preparative TLC was done on Silica gel HF254 plates with
mobile phase chloroform: methanol: ­NH4-water:: 55:75:5
(De Faria et al. 2011) and bands were observed under UV
light. The bands were purified by scraping the individual
TLC bands and dissolving in methanol followed by filtra-
tion using 0.2 µm syringe filter. The purity of the bands was
further checked in analytical TLC.
Characterization
The purified BS was then characterized by analytical TLC,
LCMS and FTIR spectroscopy. For FTIR, the purified BS
sample was dissolved in dichloromethane and subjected to
Page 3 of 12 297
IR scanning from 700–4000 cm−1 in the FTIR spectropho-
tometer (Alpha FTIR, Bruker, Germany) with the resolu-
tion of 4 cm−1 and the instrument was equipped with Opus
graph plotter (Pornsunthorntawee et al. 2008). For further
characterization, the NMR technique was used and 24 µg/
ml purified BS sample was dissolved in deuterated metha-
nol-d4 solution. 1H-NMR analyses were done with Bruker
AFCEND-Germany spectrophotometer with the frequency
of 400 megahertz (Makkar and Cameotra 1999). The spec-
trograph was studied with analytical chemistry software
‘Mestrenova’. Finally, LCMS was used to determine the
molecular mass of the biosurfactant. 10 µg/ml purified BS
was dissolved in HPLC grade methanol and applied for ESI
& APCI MS (Thermo Finnigan LCQ Advantage Max Ion
Trap Mass Spectrometer Hyphenated with Thermo Finnigan
Surveyor HPLC system) with the mass range of 50–2000 Th.
The spectrograph was studied with an analytical chemistry
software ‘Mestrenova’ (Antonious et al. 2015).
Effect of BS on corn seed germination
Seeds were sterilized with 3% sodium hypochloride solution
and were washed thoroughly with distilled water. Purified
BS solutions of 0, 150, 300, 450 and 600 µg/ ml concentra-
tions were prepared. Blotting paper (Whatman No. 1) was
placed at the bottom of 90 mm Petri plates and was soaked
with 10 ml of BS solutions. Control plates were soaked
with 10 ml sterilized distilled water. Five seeds were placed
in each plate and incubated in a dark place at room tem-
perature. Seed germination and growth of seedlings were
observed at the regular interval of 24 h for one week.
Effect of BS producing bacteria on corn plant growth
The previous experiment showed the growth-promoting
property of BS in the seedlings of corn. Earlier literatures
have also reported that rhizobacteria produce several organic
compounds including BS that enhance the plant growth
(Okoliegbe and Agarry 2012; Sachdev and Cameotra 2013).
It motivated us to study the effect of biosurfactant producing
bacteria on plant growth. The hypothesis behind the experi-
ment is that the bacteria produce the BS in the rhizosphere
that enhances the plant growth as described in the seed ger-
mination experiment (2.4).
Preparation of Pot microcosm
The soil was collected randomly from the maize growing
agricultural land of Sumbuk, Sikkim, India, and was steri-
lized twice at 121 °C for 20 min. Ten pots were filled with
1.25 kg of soil each. The cell density of the 48 h old Bacillus
tequilensis was maintained at ­OD600 of 0.08 in UV–Visible
spectrophotometer (PerkinElmer Lambda 25) which was
13
297 Page 4 of 12
equivalent to ­108 cells/ml (Bai et al. 2003; Bhuvaneswari
et al. 1980). The five sets of pots were inoculated with 20 ml
of broth culture with the density of the inoculum of 8 × 105
cells ­kg−1 and incubated for 7 days at room temperature. A
controlled set of pots were given 20 ml of sterile nutrient
broth. Both the set of pots were not treated with any kind of
fertilizer or manure instead, pots were sprinkled with water
for 1 week to activate the microorganisms.
Sampling regime and analytical methods
After 1 week, the maize seeds “Vivek Maize Hybrid 53”
were sown in both sets of the above-mentioned pots. The
plant growth was determined on the basis of the number of
days of seed germination, growth in plant height, number
of leaves and leaf length. The fresh weight, dry weight and
moisture content were estimated at the end of the experi-
ment. The moisture content of the maize plants was analyzed
by automated moisture analyzer (Ohaus/MB-35, United
States).
After five weeks, when the experimental plants were
harvested, the dry weight and moisture analyses of the pot-
ting soil were conducted. The population of Bacillus sp.
was determined in the treated soil and control soil by serial
dilution method. Mycorrhiza spores were determined from
both sets of 100 g of soil by the sieving method. Mycorrhiza
spores were counted under 50× with the help of a compound
microscope.
The data were analyzed using STATA (Software for Sta-
tistics and Data Science) and XLSTAT (Microsoft). The dif-
ference in the means of the data was analysed by analysis of
variance (ANOVA). Differences with probabilities of 0.05
or lower were considered as significant.
Results
Isolation and screening of biosurfactant and strain
selection
57 bacterial isolates were obtained from eight kinema sam-
ples. Based on cell morphology, Grams’ staining and pres-
ence of spore, the isolates were tentatively identified as
Bacillus isolates. All these isolates were screened for BS
production.
Out of 57 Bacillus isolates, only ten were found posi-
tive in initial screening (Supplementary Table 1). Bacillus
isolates LK5.4 showed the maximum oil displacement zone
of 3 cm. Maximum emulsification index ( EI 24 = 52.31) was
observed in the BS sample of Bacillus isolate LK5.4 with
sunflower oil. It suggested that the BS produced by this iso-
late had good emulsification efficiency. Similarly, this isolate
could reduce the surface tension of the culture broth by more
13
3 Biotech (2020) 10:297
than 40%. BS produced by this isolate was 759 mg L−1 on
dry weight basis which was the highest among other isolates.
Thus, it can be concluded that Bacillus LK5.4 isolate has the
potential to produce a relatively high quantity of BS.
The identity of these potential biosurfactant producers
was determined on the basis of different phenotypic and
genotypic characteristics. Result of the microscopic and
biochemical tests indicated the isolate LK5.4 as Bacillus
sp. (Table 1). Further, the similarity search of 16S rRNA
gene sequence of LK5.4 with the help of NCBI BLAST tool
confirmed its identity as Bacillus tequilensis. The sequence
was submitted to the NCBI GeneBank with the accession
number KY083699. Consensus neighbour-joining tree was
constructed using ‘Molecular Evolutionary Genetic Analy-
ses’ (MEGA) software version 10.0.5 (Fig. 1).
Characterization of biosurfactant
Initially, the analytical TLC was used to determine the bio-
chemical nature of the biosurfactant. TLC chromatogram
upon treatment with ninhydrin showed pink spots that indi-
cated the lipopeptide nature of the BS. Three major spots
on the TLC plates were observed with the Rf value of 0.78,
0.83 suggesting surfactin or some isotypes of surfactin (Cmn
et al. 2017; Thaniyavarn et al. 2003) and the third band with
Rf 0.98. These bands also showed blue fluorescence on pre-
parative TLC plates with silica H­ F254 under U­ V322 (Fig. 2).
The LCMS was used to identify the BS on the basis of m/z
nature. The results of the LCMS are presented in Fig. 3. The
primary evidence for the identification of BS form Bacillus
tequilensis LK5.4 showed the MS peaks at m/z 1086, which
corresponds to [M + Na]+ C17 surfactin (Sarwar et al. 2018).
Another peak at m/z 1081 also showed a variant of sodiated
molecule of surfactin (Ma et al. 2016). The next peak at m/z
1072 indicated the presence of sodium associated C15 sur-
factin (Perez et al. 2017). A characteristic peak at m/z 1065
for iturin was also observed (Dimkic et al. 2017). The peak
at m/z 1058 showed another variant of [M + Na]+ surfactin
(Pecci et al. 2010). Remaining peaks at m/z 1050 indicated
the surfactin with the amino acid chain Glu-Leu/Ile-Leu-
Leu/Ile-Asp-Leu-Val associated with β-hydroxy C16 fatty
acid chain (Ma et al. 2016). The peak at m/z 1044 also cor-
responds to surfactin (Pecci et al. 2010). Peaks at m/z 1036
and 1030 for surfactin were also reported (Chen et al. 2017).
The peak at m/z 1022 and 1008 also confirmed the presence
of surfactin (Sarwar et al. 2018).
FTIR spectroscopy was also used to evaluate the struc-
tural characteristics of purified BS. Infrared spectroscopy
(Fig. 4) showed the absorbance in the region of 2855 to
2924 cm−1 which corresponds to carbon–carbon (C–C)
stretching and absorbance at 1456 cm−1 showed carbon-
hydrogen (C=H) double bond stretching (Silverstein et al.
2005). Absorbance at these regions indicated the presence of
3 Biotech (2020) 10:297 Page 5 of 12 297

lipopeptide moiety in the structure. CO–N bonds and N–H

Casin Gelitin Starch Arabinose Glucose Mannose Mannitol Salicin Starch Xylose
deformation in combination with C–N was also observed

+
at 1648 cm−1 and 1542 cm−1 respectively (Silverstein et al.
2005) suggesting the presence of amino acid in the structure.
It also showed the broad peak of –OH at 3352 cm−1 which

−
may be because of residual water present in the sample or
–OH bonds of the lipopeptide chain (Silverstein et al. 2005).

+
NMR also added the information and confirmed the struc-
tural characteristics of BS. The BS isolated from B. tequilen-
sis LK5.4 showed triplet peak at 0.8 ppm which indicated

+
the presence of terminal –CH3 of the fatty acid chain while
Sugar Fermentation

duplet of peaks at 1.2 ppm, 1.49 ppm, 1.85 ppm indicated

the non-saturated hydrocarbon chain of fatty acid (Fig. 5).
+

The peak at 2.55 ppm showed –CH 2–COO −, duplet at

3.0 ppm showed the presence of –O–CH–, peak at 4.35 ppm
+

showed a terminal group of the amino acid (Balan et al.

2017). Some peaks were also found in the region of 6.6, 7.0
and 7.5 ppm indicating the presence of the aryl group. The
peak at 8.3 ppm showed the presence of terminal amide that
−

confers the presence of amino acid in the BS (Tiwary and

Dubey 2018).
Table 1  Morphological and physiological characteristics of biosurfactant producing Bacillus isolate LK 5.4 from kinema

+
Hydrolysis of

Effect of biosurfactant on corn seed germination

+

Observation of corn seed germination in the petri-plates

+

treated with different concentrations of BS showed the rate

Biochemical characteristics

of germination was maximum at 300 µg/ml compared to the

utilization

untreated control (p < 0.05) (Fig. 6).

Citrate

Effect of biosurfactant producing B. tequilensis

Proskauer

LK5.4 on maize plant

Voges–

A significant difference (p < 0.05) in the plant growth was

+ for the positive result and − for the negative result for the respective test
The isolates was Gram’s positive, endospore producing, catalase positive
10 20–50 65 5–10

observed during 5 weeks experiment (Fig. 7). First, the

pH

influence of B. tequilensis in the seed germination was

observed. Treated plants showed a mean germination time
−
NaCl conc Temperature

of 4.4 days while in control plants the mean germination

time was 8 days (Fig. 7a). The mean height of treated plants
+
(°C)

was 29.4 cm; whereas in control plants it was 15.48 cm

−
Growth in/at

(Fig. 8). No significant difference in the number of leaves

was observed in 5 weeks as the mean of the number of leaves
Position of 2–10%

in treated plants was 3.4 and in case of control, it was 3.

+

The average length of leaves in treated plants was 14.17 cm,

on the other hand in control plants, the average length was
endospore

Cylindrical Central

11.12 cm (Fig. 7b). The mean fresh weight of the 5 treated

plants was 1.125 mg and the mean of fresh weight in con-
trol plants was 0.586 mg (Fig. 7c). In the same way, the dry
Microscopy

endospore
Shape of

weight and moisture content in treated plants was 0.463 mg

and 68.48% respectively. The dry weight and moisture con-
tent in control plants was 0.222 mg and 50.53%, respectively
(Fig. 7c, d). These results indicated that the BS producing
LK.5.4

Bacillus LK5.4 play an important role in increasing biomass

Isolate
code

of the plant.

13
297 Page 6 of 12 3 Biotech (2020) 10:297

Fig. 1  Evolutionary relation-

ship of taxa. The evolutionary
history was inferred using the
Neighbor-Joining ­method22. The
optimal tree with the sum of
branch length = 0.61495206 is
shown. The percentage of rep-
licate trees in which the associ-
ated taxa clustered together
in the bootstrap test (500
replicates) are shown next to the
­branches23. The tree is drawn
to scale, with branch lengths in
the same units as those of the
evolutionary distances used to
infer the phylogenetic tree. The
evolutionary distances were
computed using the Jukes-
Cantor ­method24. Evolutionary
analyses were conducted in
­MEGA725

Fig. 2  Analytical TLC plate of spores in the treated soil was a 640 kg−1 soil, while in the
BS observed under UV light at control pots mean mycorrhiza spores count was 235.6 kg−1
322 nm soil (Fig. 10). This clearly showed that the bacteria inocu-
lated in the soil supports the growth of mycorrhiza.

Discussion

According to Allied Market Research, the global market of

Determination of microbial load in the control and treated
plants at the end of the experiment showed that the microbial
load was significantly higher (p < 0.05) in treated pots (7.78
log cfu g­ −1) compared to that in the control pots (6.75 log
cfu ­g−1) (Fig. 9). Similarly, the mean number of mycorrhizal
surfactant was 43,666 million US Dollars in 2017 and this
has been predicted to reach 66,408 US Dollars within seven
years because of its application in household detergents,
personal care, food processing, oil field chemicals, agricul-
tural chemicals and so on (Shastri and Sumant 2018). How-
ever, most of the surfactants are chemically synthesized and
contribute to soil salinity as well as soil pollution (Eugenio
et al. 2018). While most of the BS has been studied from
microorganisms from the petroleum-contaminated soil and
water (Bento et al. 2005; Joshi et al. 2013; Saravanan and
Vijayakumar 2012; Sim et al. 1997), this study is focused
on the BS from Bacillus sp of kinema and its application
in maize cultivation. The BS producing Bacillus from kin-
ema, till now has remained unexplored for this kind of study.
Kinema is a soybean fermented food with high amount of
proteins (47.3–48%), fats (16.1–17.7%) and free fatty acids
(2.4–3.0%) (Sarkar et al. 1994) which are used as a major
precursor for the biosynthesis of lipopeptide BSs in Bacil-
lus sp. (Zhi et al. 2017). The common species reported in
kinema for the fermentation of soybean are B. cereus, B.
circulans, B. licheniformis, B. sphaericus, B. subtilis and
B. thuringiensis (Sarkar et al. 2002). We did not find any

13
3 Biotech (2020) 10:297
Fig. 3  ES-MS spectrum of biosurfactant from B. tequilensis LK5.4 showed peaks at m/z 1086,1072, 1058 and 1044 for [M + Na]+ surfactin C17,
C16, C15 and C14, respectively
Fig. 4  FTIR spectrum of TLC purified biosurfactant from B. teq-
uilensis LK5.4
previous report that mentions the presence of B. tequilensis
from kinema although the best BS producing isolate LK5.4
was identified as B. tequilensis based on 16S rRNA gene
sequencing.
Page 7 of 12 297
According to Romero et al. (2016) the Rf values of iturin,
surfactin and fengycin were 0.3, 0.7 and 0.09, respectively
in analytical TLC (Romero et al. 2007). Arrebola (2009)
reported the Rf value of fengycin, iturin A and surfactin
were 0.2, 0.6 and 0.75, respectively (Arrebola et al. 2009).
Rf values of BS vary slightly with the modification of the
mobile phase and the solvent system in which its solution
is prepared. Therefore, the BS having the Rf value close to
0.7 or 0.75 can either be surfactin or its isotope (Cmn et al.
2017; Thaniyavarn et al. 2003). Based on TLC analysis, the
BS isolated from B. tequiiilensis LK5.4 was tentatively iden-
tified as surfactin. Perez et al. (2017) reported MS peaks in
association with ­H+, ­Na+, and ­K+ at 1036, 1058 and 1074
as surfactin A with C-15, whereas the peaks at m/z 1050,
1072 and 1088 as surfactin A with C-16 and peaks at m/z
1065, 1086 showed the presence of surfactin A with C-17
(Perez et al. 2017). The peak at m/z 1022 also corresponds
to surfactin (Chen et al. 2017). LCMS confirmed the iden-
tity of the BS and proved that the sample contains different
isotypes of the surfactin. Peaks for amino group, carboxyl
group, hydroxyl group and C–H bond, C=H bonds C–N
bond, C–O bonds have been confirmed through FTIR and
NMR analysis which is in agreement with the data of MS
and TLC analysis.
13
297 Page 8 of 12
Fig. 5  H1 NMR spectra of TLC purified biosurfactant from B. teq-
uilensis LK5.4 dissolved in deuterated methanol-d4 solution. Peaks in
the range of 1.2 to 1.8 ppm hydrocarbon chains belong to fatty acid
Fig. 6  Effect of different concentrations of biosurfactant on the
growth of seedlings. Data mean of 10 replicates and the vertical bar
represents the standard error
13
3 Biotech (2020) 10:297
and peaks at 2.5 ppm for the acidic group, however, peak at 4.3 indi-
cates amino group (strong evidence of amino acid)
Indeed a lot of work has been done on the application
of surfactin in the last few decades but most of the work is
focused on petroleum extraction and biodegradation (Dim-
kic et al. 2017; Varadavenkatesan and Murty 2013). Few
review articles also claim that the BS has great potential
to enhance the plant growth (Okoliegbe and Agarry 2012;
Sachdev and Cameotra 2013). While other studies have
shown that the surfactin and some other BS have biologi-
cal control activity against many plant pathogens (Ongena
and Jacques 2007; Perez et al. 2017). In this work, the
effect of BS was observed on seedling germination under
in-vitro condition, which was found most effective at the
concentration of 300 μg/ml. These BS can be used directly
as fertilizer on agriculture land, but it will be expensive
because of the high cost of production and downstream
processing. Indeed the BS producing B. tequilensis LK5.4
showed a significant effect on the growth of the maize
plants and the direct use of this isolate as biofertilizer can
be a cost-effective tool for crop improvement. Bacteria
produce several organic acids that solubilize the phospho-
rus and other minerals (Anastasio et al. 2010) and bio-
surfactant can increase the bioadsorption of the minerals
(Sachdev and Cameotra 2013). Moreover, soil analysis
3 Biotech (2020) 10:297 Page 9 of 12 297

Fig. 7  Effect of biosurfactant producing B. tequilensis LK5.4 on ger- A = Fresh weight, B = Dry weight, d effect on Moisture content of the
mination and growth of corn plants. Control, Treated. a Effect plant. Data were collected for one month. Data mean of five repli-
on seed germination, b effect on leaf length, c effect on plant weight cates, the vertical bar represents standard error

Fig. 9  Effect of B. tequlensis LK5.4 on bacterial load in the potted

Fig. 8  Effect of B. tequilensis LK5.4 on the height of corn plants soil after harvesting the corn plants were Control, Treated. The
were Control, Treated. Data mean of five replicates Data represents the mean of five replicates and the vertical bar repre-
and vertical bars represents the standard errors sents the standard error

13
297 Page 10 of 12
Fig. 10  Effect of B. tequilensis LK5.4 on mycorrhizal load in potted
soil after harvesting the corn plants were Control, Treated.
Data represent the mean of five replicates and the vertical bar repre-
sents the standard error
showed that the direct inoculation of B. tequilensis LK5.4
resulted in a higher load of Bacillus sp. in the treated soil
compared to that in the control set. Members of Bacillus sp
such as B. luciferensis, B. amyloliquefaciens and B. subti-
lis have been found to enhance the growth of the plants by
producing growth promoters and antagonistic properties
(Panneerselvam et al. 2019).
Conclusion
The present work documented the BS producing B. teq-
uilensis LK5.4 from kinema, the ethnic fermented soybean
product of Sikkim. This is the first report of B. tequilensis
isolated from fermented food with potential for production
of surfactin. Surfactin produced by LK5.4 showed a promis-
ing effect on seed germination. Besides, B. tequilensis LK5.4
demonstrated a significant plant growth-promoting activity
which indicated its potential to be developed as a bioferti-
lizer to augment the organic farming in the region.
Acknowledgements Authors acknowledge Sikkim University for
providing UGC Non-NET fellowship to Lalit Kumar Chaurasia to
carry out this research. Authors are also thankful to Dr Rajesh Kumar
Khulbe, Senior Scientist, ICAR-Vivekanand Parvatiya Krishi Anusand-
han Sansthan, Almora, India, for providing “Vivek Maize Hybrid 53”
seeds for the experiments.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of in-
terest. This article does not contain any study with human participants
and animals performed by any of the authors.
13
3 Biotech (2020) 10:297
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