Biotransformation of benzoin by Sphingomonas sp. LK11 and ameliorative effects on growth of Cucumis sativus

Article abstract of DOI:10.1007/s00203-019-01623-1

Plant endophytes play vital role in plant growth promotion as well as in abiotic and biotic stress tolerance. They also mediate biotransformation of complex organic materials to simpler and useful by-product. Therefore, the role of plant endophyte in plant growth promotion and stress tolerance has gained considerable attention in recent days. Sphingomonas sp. LK11 is an important plant endophyte that actively regulates plant growth. However, the biotransformation and stress tolerance potential of Sphingomonas sp. LK11 was yet to be elucidated. Therefore, we studied the biotransformation of benzoin by Sphingomonas sp. LK11. We found that, Sphingomonans sp. LK11 biotransformed benzoin to benzamide. Further application of benzamide to Cucumis sativus led to decrease in agronomic potential of C. sativus as benzamide acts as an abiotic stress agent. However, the application of Sphingomonas sp. LK11 inoculums with benzamide reverted back the agronomic trait of the plants, suggesting the role of Sphingomonas sp. LK11 in biotransformation and abiotic stress tolerance in plants.

Full text of DOI:10.1007/s00203-019-01623-1

Archives of Microbiology
https://doi.org/10.1007/s00203-019-01623-1
ORIGINAL PAPER
Biotransformation of benzoin by Sphingomonas sp. LK11
and ameliorative effects on growth of Cucumis sativus
Amjad Ali1 · Tapan Kumar Mohanta1 · Sajjad Asaf1 · Najeebur Rehman1 · Saif Al‑Housni1 · Ahmed Al‑Harrasi1 ·
Abdul Latif Khan1 · Ahmed Al‑Rawahi1
Received: 19 September 2018 / Revised: 14 December 2018 / Accepted: 22 January 2019
© Springer-Verlag GmbH Germany, part of Springer Nature 2019
Abstract
Plant endophytes play vital role in plant growth promotion as well as in abiotic and biotic stress tolerance. They also medi-
ate biotransformation of complex organic materials to simpler and useful by-product. Therefore, the role of plant endophyte
in plant growth promotion and stress tolerance has gained considerable attention in recent days. Sphingomonas sp. LK11
is an important plant endophyte that actively regulates plant growth. However, the biotransformation and stress tolerance
potential of Sphingomonas sp. LK11 was yet to be elucidated. Therefore, we studied the biotransformation of benzoin by
Sphingomonas sp. LK11. We found that, Sphingomonans sp. LK11 biotransformed benzoin to benzamide. Further applica-
tion of benzamide to Cucumis sativus led to decrease in agronomic potential of C. sativus as benzamide acts as an abiotic
stress agent. However, the application of Sphingomonas sp. LK11 inoculums with benzamide reverted back the agronomic
trait of the plants, suggesting the role of Sphingomonas sp. LK11 in biotransformation and abiotic stress tolerance in plants.
Keywords Sphingomonas sp. LK11 · Biotransformation · Benzoin · Benzamide · Abiotic stress
Introduction
phosphate that contribute significantly towards the growth
and stress tolerance in plants (Kumar et al. 2014; Etesami
et al. 2015; Oteino et al. 2015; Joe et al. 2016; Khan et al.
2016; Ghavami et al. 2016). Few bacterial endophytes
carry out biological nitrogen fixation and a few helps for
bioconversion of organic molecules. Therefore, bacterial
endophytes play diverse roles towards the growth, develop-
ment, and stress responses in plants (Hardoim et al. 2008;
Dai et al. 2016; Santoyo et al. 2016). Current studies have
investigated the potential of endophytic bacteria in biodeg-
radation as well that converts organic pollutants to beneficial
chemicals in soil (Stępniewska and Kuźniar 2013; Zhang
et al. 2014; Zhu et al. 2016; Iqbal et al. 2017). The majori-
ties of these study have been focused on bacteria of plant
rhizosphere origin. There are 4000,000 plant species exist-
ing on earth and nearly all of the plant species are colonized
by the endophytes without any sign of infection (Mohanta
2013). However, only a few plant species have been studied
relative to their endophytes. Therefore, it is highly important
to find novel and beneficial endophytes from diverse plant
communities. Bacterial endophytes possess advantage over
the bacteria inhabiting in the rhizosphere (Santoyo et al.
2016). Living within the host tissues or cell enables direct
opportunity to remain in contact to exert a direct beneficial
The endosphere of the plant contains diverse microbial com-
munities and contributes significantly towards the growth,
development, and stress responses (Ryan et al. 2008; Bae
et al. 2016; Santoyo et al. 2016; Ullah et al. 2017; Hassan
2017). Bacteria inhabit the plant endosphere during its life
cycle without causing any harm to the host plant. Coloniza-
tion of endophytic bacteria leads to the increase in plant
biomass and height (Mei and Flinn 2010; Knoth et al. 2014).
Bacterial endophytes synthesize plant hormone including
indole-3-acetic acid, secrete siderophores, and solubilize
Communicated by Erko Stackebrandt.
Amjad Ali and Tapan Kumar Mohanta contributed equally.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00203-019-01623-1) contains
supplementary material, which is available to authorized users.
* Tapan Kumar Mohanta
nostoc.tapan@gmail.com
1
Natural and Medicinal Plant Research Center, University
of Nizwa, Nizwa 616, Oman
Vol.:(0123456789)
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effect. Therefore, the beneficial roles of plant endophytes are
enormous and hence they can be used for diverse beneficial
purposes. Previously, we had isolated endophytic bacteria,
Sphingomonas sp. LK11 from the leaves of arid medicinal
plants (Tephrosia apollinea). We found, Sphingomonas sp.
LK11 actively increases the growth of the plant biomass.
In addition, the endophyte also helps to overcome salin-
ity and cadmium stress in tomato (Khan et al. 2014; Halo
et al. 2015). Our previous finding suggests that this bacterial
strain was able to produce phytohormones including auxin
and gibberellins that greatly contribute towards the growth
and development of the plant. Therefore, we conducted the
study to understand the potential growth regulatory role of
Sphingomonas sp. LK11 in cucumber plant. In addition, we
also studied the bioconversion potential of Sphingomonas
sp. LK11 from benzoin to benzamide.
Materials and methods
Screening experiment
Different hydrocarbons were checked for the solubility in
water, ethanol, and ethanol+water. Most of the hydrocarbons
were dissolved in ethanol. All the hydrocarbons with 10 mg
concentration were dissolved in 1 ml ethanol and added in
50 ml flask of sodium phosphate buffer (5 gm NaCl, 5 gm
K2HPO4, 0.75 MgSO4) with LK11. After the growth of
Sphingomonas sp. LK11 in culture filtrate, it was separated
and checked on TLC to select the desired hydrocarbon for
further experiment. Rescreening experiment was performed
on a large-scale basis that contained 100 mg of benzamide in
500 µl flask. After the growth of LK11 strain, thin layer chro-
matography (TLC) analysis was conducted. The mass of ben-
zamide was analyzed using Q-TOF liquid chromatography
mass spectroscopy [benzamide as white amorphous solid;
Q-TOF LC/MS=122.0609 [M+H]+, (calculated=122.0526,
C6H5CONH2)] (Supplementary data 1). Subsequently, it was
confirmed using 1H-NMR [CDCl3, (600 MHz): δ 7.80 (2H,
t = 7.2, 1.2 Hz, H-2/6), 7.51 (1H, t = 7.8, 1.2, H-4), 7.43
(2H, t=7.8 Hz, H-3/5), 6.20 (2H, br.s, NH2)] and 13C-NMR
[(CDCl3, 125 MHz): δ169.6 (C=O), 133.3 (C-1), 132.0
(C-4), 128.6 (C-2/6), 127.3 (C-3/5)] (Supplementary data 2).
Sphingomonas species has been reported mostly for
their role in bioremediation or degrading various kinds of
organic and inorganic pollutants from diverse contamina-
tion sites. Functional analysis of Sphingomonas sp. LK11
genome has reported the genes responsible for nitrate/nitrite
ammonification, and phosphate solubilization (pstC, pstA,
phoU, phoQ, nad(P), phoR (sphS), phoB, pstB, oprO, pstS)
(Asaf et al. 2018). The genome of Sphingomonas sp. LK11
encoded 3656 protein coding genes (Asaf et al. 2018). It also
possess the ability to accumulate intracellular Zn2+, reduce
Cd2+ uptake and increase the expression of metallothionein
that seclude heavy metals and their binding by their related
proteins (Khan et al. 2014) The endophyte has the poten-
tial to grow productively in salinity stress without using its
active cellular assets for the production of antioxidant and
related enzymes including peroxidase (POX), polyphenol
oxidase (PPO) and catalase (CAT) (Halo et al. 2015). Some
of the recent studies have reported that, Sphingomonas sp.
can degrade the organic compound bisphenol (Fujiwara et al.
2016), phenol (Gong et al. 2016), triclocarban (Mulla et al.
2016), phenanthrene(Liu et al. 2016), chlorogenic acid (Ma
et al. 2016), nonylphenol polyethoxylates (Bai et al. 2018),
astaxanthin (Ma et al. 2016), dioxin (Miller et al. 2010),
γ-hexachlorocyclohexane (Tabata et al. 2013), nicotine (Zhu
et al. 2016), plasticizer (Kera et al. 2016), and hexachlo-
rocyclohexane isomers (Kumari et al. 2002). Previously,
our group has reported that LK11 has the potential to be
used as a plant growth-promoting bacteria. Considering all
these useful characteristics of Sphingomonas sp. LK11, we
proposed to investigate the biotransformation potential of
benzoin organic compound. Further, we studied the role of
biotransformed compound of benzoin towards plant growth,
development, and stress tolerance.
Bacterial growth and OD in hydrocarbons
The bacterial endophyte (LK11) was grown in sodium phos-
phate buffer (5 gm NaCl, 5 gm K2HPO4, 0.75 gm MgSO4)
for 24 h with continuous shaking. Post 24 h, OD of the strain
was checked at 48, 96, and 120 h, respectively.
Plant growth condition
The current study used C. sativus plants. Briefly, seeds were
surface-sterilized with NaOCl (5% v/v) for 10 min and thor-
oughly rinsed with autoclaved distilled water. Seeds were
sown in plastic pots containing horticulture soil [peat moss
(13–18%), perlite (7–11%), coco-peat (63–68%) and zeolite
(6–8%), while the macro-nutrients present were as follows:
NH4+ ~90 mg/L, NO3− ~205 mg/L, P2O5 ~350 mg/L, and
K2O~100 mg/L] under controlled greenhouse conditions at
temperatures of 30 2 °C (Kang et al. 2014).
Inoculation of LK11 with Benzamide
C. sativus plants were grown in pots and inoculated with
LK11 endophyte strain. Inoculation was done 3 times after
every 3 days. Similarly, the plants treated with benzamide
were also inoculated with endophytes LK11 and inoculation
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was done 3 times after every 3 days. The control plants were
grown under normal condition in soil pots after treatment, the
plants were left to grow for 28 days. Post 28 days of treatment,
plants were harvested and stored in − 80 °C for further use.
absorbance of the purpurogallin formed was taken at 420 nm.
One unit of peroxidase and polyphenol oxidase was defined as
an increase of 0.1 units of absorbance.
Reduced glutathione (GSH) content was measured accord-
ing to the method of Ellman (1959). Briefly, samples were
ground in a chilled mortar and pestle using 3 ml of 5% (v/v)
trichloroacetic acid. The homogenates were centrifuged at
10,000×g for 15 min at 4 °C. The supernatant (0.1 ml) was
added to 3.0 ml of 150 mM NaH2PO4 (pH 7.4). 500 µl of
5,5′-dithio-bis(2-nitrobenzoic) (DTNB) (75.3 mg of DTNB
was dissolved in 30 ml of 100 mM phosphate buffer, pH 6.8)
was later added. The mixture was incubated at 30 °C for 5 min.
Absorbance was determined at 412 nm and GSH concentration
was calculated by comparing it to a standard curve. All the
experiments were repeated thrice.
Plant growth and root analysis
Plant growth data were taken for all the treated and control
samples. The growth parameters considered for analysis dur-
ing this experiment were plant height (cm), fresh and dry
weight of shoot and roots (gm). The data were collected
from ten plants for each experiment where each experiment
was replicated three times.
Superoxide anion was detected by the method as described
by Doke (1983). This assay is depends on its ability to reduce
nitro blue tetrazolium (NBT). Using this method, the ground
leaves were immersed in 10 mM phosphate buffer, pH 7.8,
containing 0.05% (w/v) NBT and 10 mM NaN3 in 2 ml Eppen-
dorf. Subsequently, phosphate buffer was added, and the reac-
tion was incubated at room temperature for. After incubation,
the sample was heated at 85 2 °C for 15 min and cooled
down immediately. Optical density was measured at 580 nm,
and the O2− content was expressed as an increase in absorb-
ance/0.1 g dry weight.
Antioxidant’s analysis of cucumber plants
The ground leaf powder was immersed in protein extraction
buffer for quantification of protein content. Later, the sam-
ples were added with 50 mMTris-HCl buffers (pH 7.0) con-
taining 3 mM MgCl2 1 mM EDTA, and 1.0% PVP. Subse-
quently, the samples were centrifuged at 2500×g for 15 min
at 4 °C. The supernatant was collected for further study.
The absorbance of the protein samples was measured at
260 nm by adding Bradford reagents with extraction buffer.
The samples were vortexed and centrifuged to separate the
supernatant. For determination of protein content, the super-
natant was used for biochemical analysis. All parameters
were expressed as activity per milligram protein.
Lipid peroxidation activity was determined by the method
as described by Ohkawa et al. (1979). In this method we
added, 0.2 ml 8.1% sodium dodecyl sulfate, 1.5 ml of 20%
acetic acid (pH 3.5), and 1.5 ml of 0.81% thiobarbituric acid
aqueous solution was added in succession in a reaction tube.
This step was followed by addition of 0.2 ml of tissue homoge-
nate extracted with 10 mM phosphate buffer (pH 7.0). Later,
samples were boiled and cooled down the reaction mixture
to room temperature followed by addition of 5 ml butanol/
pyridine (15:1 v/v). The optical density of the samples was
measured using spectrophotometer at 532 nm. Tetramethoxy-
propane was used as an external standard.
Catalase (E.C1.11.1.6) activity was assayed by the method
of Aebi (1984). The crude enzyme extract was treated with
0.5 ml of 0.2 M H2O2 in 10 mM potassium phosphate buffer
(pH 7.0). Catalase activity was estimated by decrease in
absorbance of H2O2 at 240 nm and one unit of catalase was
defined as µg of H2O2 released per mg protein per min−1.
The activities of Peroxidase (E.C 1.11.1.7) and polyphe-
nol oxidase (E.C 1.10.3.1) were measured as described by
Kar and Mishra (1976) with minor modifications (Kar and
Mishra 1976). The samples were homogenized with potas-
sium phosphate buffer pH 6.8 (0.1 M) and centrifuged at 2 °C
for 15 min at 5000×g. The assay mixture for the peroxidase
activity comprised 0.1M potassium phosphate buffer (pH 6.8),
50 µl pyrogallol (50 µM), 50 µl of H2O2 (50 µM), and 0.1 ml
enzyme extract. The mixture was incubated for 5 min at 25 °C
followed by addition of 0.5 ml 5% (v/v) H2SO4 to terminate the
reaction. The amount of purpurogallin formed was determined
by absorbance at 420 nm. The same assay mixture without
H2O2 was used to assay the activity of polyphenoloxidase. The
Determination of photosynthetic pigments
The chlorophyll and carotenoid content were determined as
described by Lichtenthaler and Wellburn (1983). The leaves
of cucumber plant were ground in presence of 80% acetone
to determine the photosynthetic pigment. The absorbance of
photosynthetic pigment is different for chlorophyll a (663 nm),
b (645 nm), and carotenoid (470 nm). Chlorophyll content was
calculated using the following formulae:
[
]
Chlorophyll a(mg∕g FW) = {(12.7 × A663) − (2.69 × A645)}∕1000 × W × V
[
]
Chlorophyll b(mg∕g FW) = {(22.9 × A645) − (4.68 × A663)}∕1000 × W × V
Total carotenoid content = (1000A470 − 1.82chlorophyll a 85.02chlorophyll b)∕198
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where W is the fresh weight and V is the extraction volume.
Oxidative stress regulation by Sphingomonas sp.
LK11 during benzamide contamination
Phylogenetic analysis
The overall results showed that LK11 inoculation signifi-
cantly improve the plant growth and development during
benzamide stress. Although, the overall morphological
data were significantly different in response to Sphingo-
monas sp. LK11 inoculation to plants, we further assessed
the physiological responses of the cucumber plants against
plant–microbe interaction during metal contamination under
short and long-term. In this regard, total protein, catalase,
peroxidase, polyphenol oxidase, reduced glutathione, super-
oxide anion, and lipid peroxidation was quantified among
different samples and treatments (Fig. 1).
Before construction of the phylogenetic tree, the nucleotide
sequence of 16S rRNA of Sphingomonas sp. LK11 was sub-
jected to BLASTn analysis in NCBI (National Center for
Biotechnology Information) using bacterial reference data-
base. The BLASTn resulted 16S rRNA of closely related
species were downloaded and aligned using MUSCLE to
obtain a clustal file. The resulted clustal file was converted
to MEGA file format. The resulted MEGA file of 16S rRNA
was used to model selection in MEGA6 software (Tamura
et al. 2013). The model with lowest BIC value was used
to construct the phylogenetic tree. Following parameters
were used to construct the phylogenetic tree; statistical
method, maximum likelihood; number of bootstrap repli-
cations, 1000; substitution type, nucleotide; model/method,
Hasegawa-Kishino-Yano model; rates among sites, Gamma
distributed (G); gaps/missing data treatment, partial dele-
tion; site coverage cutoff, 95%; branch swap filter, very
strong.
Benzamide stress‑induced oxidative stress
mitigation by Sphingomonas sp. LK11
The content of peroxidase, catalase, and reduced glutathione
was lower in LK11-inoculated plants compared to the ben-
zamide-treated plants (Fig. 1). The peroxidase content was
significantly (p<0.009) lower in Sphingomonas sp. LK11
and Sphingomonas sp. LK11 with benzamide-treated plants
(Fig. 1). The content of polyphenol oxidase was significantly
(p < 0.0001) higher (14% and 10%) in Sphingomonas sp.
LK11 treated plants whereas Sphingomonas sp. LK11 with
benzamide-treated plants shown reduced activities, respec-
tively (Fig. 1). Similarly, catalase content was significantly
(p<0.009) lower (68%) in Sphingomonas sp. LK11 inocu-
lated plants (Fig. 1) while the reduced glutathione has no
significant difference between the inoculated and non-inoc-
ulated plants (Fig. 1).
Statistical analysis
All the samples were analyzed in triplicate. The data are pre-
sented as the mean standard error (SEM). The differences
were evaluated using two-way ANOVA. GraphPad Prism
Version 5.0 (GraphPad Software, San Diego, CA, USA) was
used for analysis of significant values at p<0.05. The mean
values were compared using Duncan’s multiple range tests
at p<0.05 (SAS 9.1, Cary, NC, USA).
Effect of benzamide stress on protein contents,
superoxide and lipid peroxidation anion in presence
of LK11
Results
Bioconversion of benzoin to benzamide
by Sphingomonas sp. LK11
No significant difference was found in total protein con-
tent between the inoculated and non-inoculated plants with
Sphingomonas sp. LK11. But inoculated plants have slightly
higher protein content than non-inoculated plants (Fig. 2).
The superoxide anion content was lower in Sphingomonas
sp. LK11 inoculated plants compared to the control. The
superoxide anions and lipid peroxidation were only signifi-
cantly lower in Sphingomonas sp. and benzamide inoculated
plants (Fig. 2).
Benzoin was treated with Sphingomonas sp. LK11 and the
reaction was allowed to continue for 6 h. The whole reaction
solution was extracted with ethyl acetate and subjected to
preparative silica gel column chromatography. The column
was eluted with 20–30% ethyl acetate/n-hexane to isolate the
benzamide with small impurities which was further purified
through preparative thin layer chromatography using 20%
ethyl acetate/n-hexane as a mobile phase. The compound
(benzamide) was previously isolated from Naravelia zey-
anica (Jaroszewski et al. 2005) and Mentha piperita (Hos-
sain et al. 2014)as well and up to the best of our knowledge,
we are the first to report the biotransformation of benzoin to
benzamide by Sphingomona sp. LK11.
Changes in chlorophyll pigments
and photosynthesis by LK11 during benzamide
stress
The chlorophyll a content was found to remain unchanged in
Sphingomonas sp. LK11 inoculated plants with and without
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Fig. 1 Effects of benzamide and LK11 on a catalase, b polyphenol oxidase, c peroxidase and d reduce glutathione
benzamide stress (Fig. 3). Chlorophyll b and carotenoid con-
tents were found to be higher in Sphingomonas sp. LK11
inoculated plants compared to the non-inoculated plants.
The chlorophyll b content was found to be significantly
(p<0.01) higher (33%) in Sphingomonas sp. LK11 inocu-
late plants compared to the control. The contents of carot-
enoid in Sphingomonas sp. LK11 and benzamide inoculated
plants showed higher (80–90%) carotenoid and chlorophyll
b content (50–60%) compared to the non-inoculated plants
(Fig. 3).
non-inoculated plants (Fig. 4; Table 1). The plants inocu-
lated with Sphingomonas sp. LK11 had highest length com-
pared to the other plants. Similarly, the fresh and dry weights
of roots were recorded. It was found that, the fresh and dry
weights of roots were higher in Sphingomonas sp. LK11
inoculated plants compared to the control. Also, the fresh
and dry weight of shoot were recorded that were higher in
inoculated plants compared to the control (Fig. 4).
Phylogenetic analysis of Sphingomonas sp. LK11
Effects of benzamide and Sphingomonas sp. LK11
on length, fresh, and dry weight of roots and shoots
A phylogenetic tree of was constructed using the 16S rRNA
of Sphingomonas sp. LK11 with the 16S rRNA sequences of
97 closely related species. Phylogenetic tree show 16S rRNA
were fall in two separate clusters and divided into three
major groups (Fig. 5). The 16S rRNA of Sphingomonas sp
LK11 was closely grouped with the 16S rRNA sequence of
Sphingomonas yabuuchi (Fig. 5). In addition, 16S rRNA of
The length of the plants was measured following benza-
mide treatment inoculated with Sphingomonas sp. LK11.
It was found that the plants inoculated with Sphingomonas
sp. LK 11 and benzamide were longer compared to the
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Fig. 2 Effects of benzamide and Sphingomonas sp. LK11 on total protein a quantification, b lipid peroxidation and c superoxide anion
Sphingomonas sp LK11 was closely grouped with the 16S
rRNA sequences of several other Sphingomonas sp. This
confirms that the studied organism belonged to genus Sphin-
gomonas. Due to unclear and physio-morphological char-
acteristics, the species information is yet to be deciphered.
deters the Lepidoptera herbivore from attacking the plants.
Therefore, we tried to understand, whether benzoin gets bio-
transformed and what would be the biotransformed prod-
uct of benzoin and its beneficial properties. In this regard’s
benzoin was biotransformed to benzamide using Sphingo-
monas sp. LK11. Benzamide is a carbonic acid amide of
benzoic acid (Asif 2015). Benzamide derivatives possess
anti-fungal activities. Therefore, the biotransformed ben-
zamide has a great potential to be used as an anti-fungal
agent against plant pathogens (Narayana et al. 2004). In
addition, benzamide is also effective against Gram nega-
tive bacteria Pseudomonas aurigenosa at 25 µg/ml mini-
mum inhibitory concentration (Asif 2015). This bacteria is
reported to be causal agent of root rot disease of Ginseng
plant (Gao et al. 2014). The conversion of benzoin to ben-
zamide and substitution of benzamide by either aliphatic,
aromatic or heteroaromatic system govern diverse biologi-
cal activities (Asif 2015). Looking all such novel activities
Discussion
In our study, we biotransformed benzoin to benzamide and
to our knowledge this is the first report regarding the bio-
transformation of benzoin to benzamide using bacterial
endophytes. Benzoin is synthesized by benzoin condensa-
tion reaction of benzaldehyde and benzoin is an important
phytoconstituent of benzoin tree (Styrax), native to warm
temperate to tropical regions. It is a dried resin exudate and
its origin most probably goes to the Middle East countries
of the Asia. This resin kills wound pathogens and hence
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Fig. 3 Effects of benzamide and LK11 on a photosynthetic pigments chlorophyll a, b chlorophyll b and c carotenoid
of benzamide, we treated this compound to understand its
role plant growth promotion. The enzymatic expression of
peroxidase, catalase, and reduced glutathione was found to
be lower in Sphingomonas-treated plants compared to the
benzamide-treated plants, suggesting the role of Sphin-
gomonas in plant growth promotion and benzamide as a
stress agent (Fig. 1). This suggests that benzamide-treated
plants show higher level of stress-related enzymes. These
stresses-related enzymes show higher level of enzyme activi-
ties, suggesting benzamide treatment has exerted significant
stress in plants. Similarly, increased level of lipid peroxida-
tion, superoxide anion, and total protein were observed in
benzamide-treated plants (Fig. 2). This suggests that ben-
zamide worked as a stress agent and hence produced higher
peroxide and superoxide ion to fight the stress. However,
when benzamide-treated plants were inoculated with endo-
phytic bacteria Sphingomonas LK11, the overall phenotypi-
cal and morphological data of the plants were shown sig-
nificantly improved. This suggests that Sphingomonas sp.
LK11 mediates oxidative stress regulation during benzamide
contamination. In benzamide-treated plants, inoculated with
Sphingomonas LK11, peroxidase, polyphenol oxidase, cata-
lase, and glutathione peroxidase contents were significantly
reduced compared to control plants. This suggests, oxidative
stress induced by benzamide is mitigated by Sphingomonas
sp. LK11 and led to growth promotion. Further, chlorophyll
a content was found to be unchanged in Sphingomonas-
inoculated plants. However, content of chlorophyll b and
carotenoids was significantly higher in Sphingomonas-
treated plants, suggesting the role of chlorophyll b in plant
growth promotion. Treatment of rhizobacteria in okra plant
leads to enhancement of the chlorophyll content as well
(Habib et al. 2016). Endophytes improve plant growth in
chick pea by enhancing chlorophyll content (Egamberdieva
et al. 2017). Enhancement of chlorophyll content in plants
is directly related to the metabolic center of the plant and
stress tolerance. Plants with higher chlorophyll content can
drastically improve the growth and development of plants
by stimulating diverse physiochemical pathways (Guru-
rani et al. 2015). Higher chlorophyll content can stimulate
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Fig. 4 Effects of benzamide and Sphingomonas sp. LK11 on fresh and dry weights of roots and shoots. Figure (a) fresh weight of shoots, b dry
weight of shoots, c fresh weight of root, and d dry weight of root
Table 1 Growth parameters of
Parameters
Control
LK11
Benzamide
LK11+benzamide
benzamide-treated plants
Plant length (cm)
19
20
17
18
Fresh weight of roots (g)
Dry weight of roots (g)
Fresh weight of shoots (g)
Dry weight of shoot (g)
5.6 0.2
0.39 0.23
13.0 0.39
1.7 0.24
6.9 0.12
0.40 0.23
18.8 0.32
2.0 0.25
6.1 0.16
0.38 0.21
13.6 0.34
2.6 0.22
6.2 0.9
0.47 0.18
16.9 0.27
2.1 0.19
higher photosynthetic rates and energy source of the plant
will be automatically increased which can greatly facilitate
growth of the plants. Higher chlorophyll b content largely
attributed to the photo system II (PS II) reaction center and
light-harvesting antenna complexes, thereby suggesting
the possible role of PS II in stress tolerance (Kalaji et al.
2016). Plants inoculated with Sphingomonas and benzamide
showed greater phenotypic characteristics compared to the
control (Table 1). Fresh and dry weight of roots and shoots
were higher in Sphingomonas and benzamide-treated plants
compared to the benzamide-treated or untreated plants. This
suggests that, Sphingomonas sp. LK11 possess the ability
to biotransform benzoin and enhances the physiological
parameters of plants under benzamide stress. The dual role
of Sphingomonas sp. LK11 is of particular interest for fur-
ther application in other plant species to exploit its biotech-
nological potential.
Hydrocarbons are ubiquitous, toxic, and persistent
organic compounds present in the environment. Micro-
bial degradation studies have shown that biodegradation
of hydrocarbon is an efficient way to remove these pollut-
ants from the environment (Kanaly and Harayama 2000).
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Archives of Microbiology
Microorganisms can produce several catabolic enzymes and
have developed different mechanisms to degrade hydrocar-
bons. The degradation of hydrocarbon is usually initiated
by hydroxylation, especially dioxygenation process, which
is catalyzed by the enzyme oxygenase (Kweon et al. 2014).
Some of the recent and previous studies have reported the
role of Sphingomonas sp. in the degradation of hydrocarbon
such as bisphenol, triclocarban, phenanthrene, nonylphenol
polyethoxylates, astaxanthin, γ-hexachlorocyclohexane,
dioxin, plasticizer, and hexachlorocyclohexane isomers and
many more (Tabata et al. 2013; Fujiwara et al. 2016; Gong
et al. 2016; Liu et al. 2016; Mulla et al. 2016). The Sphin-
gomonas sp. LK11 genome carries genes involved in the
degradation of various aromatic compounds such as benzo-
ate (AV944_13370, AV944_13375, AV944_16605), amino
benzoate, flour benzoate, quinate, naphthalene, xylene, and
toluene (AV944_02390). The beta-ketoadipate pathway,
an important bacterial energy source has been identified in
various bacteria (MacLean et al. 2006; Li et al. 2010). The
Sphingomonas sp. LK11 genome contained a gene involved
in beta-ketoadipate pathway responsible for degradation of
lignin-derived aromatic compounds. Furthermore, similar
genes involved in the degradation of various aromatic com-
pounds were reported in Sphingomonas sp. strain Ant20
as well (Ronca et al. 2015). Furthermore, Sphingomonas
sp. LK11 genome revealed the presence of genes phaB, C,
F, and R (AV944_03765, AV944_16580, AV944_14960,
AV944_16575) involved in polyhydroxybutyrate (PHB)
metabolism (Kadowaki et al. 2011). This PHB is synthe-
sized by several bacteria and stored intracellularly as gran-
ules and improves bacterial tolerance to high temperature,
H2O2 exposure, UV- irradiation, desiccation, and osmotic
stress (Ratcliff et al. 2008; Kadowaki et al. 2011). Manickam
et al. (2012) suggested that, additional supplementation of
trehalose can help in the degradation of hexachlorocyclohex-
ane (HCH) isomers by Sphingomonas sp. NM05 (Manickam
et al. 2012).
Conclusion
Sphingomonas sp. LK11 possesses the potential to biotrans-
form benzoin to benzamide. In addition, the species also
possess the ability to promote plant growth and develop-
ment through modulation of different molecular tools includ-
ing chlorophyll and antioxidant enzymes. However, it will
be of great idea to understand the genomic and molecular
details of this organism to understand the bioconversion and
growth-promotion mechanism in details.
Fig. 5 Phylogenetic tree of Sphingomonas sp. LK11 16S rRNA with 16S
rRNA of other Sphingomonas species. The phylogenetic tree was con-
structed using MEGA6 software
Author contributions AA: performed the experiments, TKM: ana-
lyzed data and drafted the manuscript, SA: performed experiments;
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Archives of Microbiology
Habib S, Kausar H, Halimi M (2016) Plant growth-promoting rhizo-
bacteria enhance salinity stress tolerance in okra through ROS-
scavenging enzymes
NR: revised the manuscript; SAH: revised the manuscript; ALK: con-
ceived the idea and revised the manuscript; ASA: conceived the idea
and revised the manuscript; AAR: revised the manuscript;
Halo BA, Khan AL, Waqas M et al (2015) Endophytic bacteria (Sphin-
gomonas sp. LK11) and gibberellin can improve Solanum lycoper-
sicum growth and oxidative stress under salinity. J Plant Interact
10:117–125. https://doi.org/10.1080/17429145.2015.1033659
Hardoim PR, van Overbeek LS, Elsas JD van (2008) Properties of bac-
terial endophytes and their proposed role in plant growth. Trends
Microbiol 16:463–471. https://doi.org/10.1016/j.tim.2008.07.008
Hassan SE-D (2017) Plant growth-promoting activities for bacterial
and fungal endophytes isolated from medicinal plant of Teu-
crium polium L. J Adv Res 8:687–695. https://doi.org/10.1016/j.
jare.2017.09.001
Compliance with ethical standards
Conflict of interest There is no competing interest to declare.
References
Asaf S, Khan AL, Khan MA et al (2018) Complete genome sequenc-
ing and analysis of endophytic Sphingomonas sp. LK11 and its
potential in plant growth. 3 Biotech 8:389. https://doi.org/10.1007/
s13205-018-1403-z
Hossain MA, Al-Hdhrami SS, Weli AM et al (2014) Isolation, fraction-
ation and identification of chemical constituents from the leaves
crude extracts of Mentha piperita L grown in Sultanate of Oman.
Asian Pac J Trop Biomed 4:S368–S372. https://doi.org/10.12980
/APJTB.4.2014C1051
Asif M (2015) Pharmacological potential of benzamide analogues and
their uses in medicinal chemistry. Mod Chem Appl 4:1000194.
https://doi.org/10.4172/2329-6798
Iqbal A, Arshad M, Hashmi I et al (2017) Biodegradation of phenol
and benzene by endophytic bacterial strains isolated from refinery
wastewater-fed Cannabis sativa. Environ Technol 1–10. https://
doi.org/10.1080/09593330.2017.1337232
Bae S-J, Mohanta TK, Chung JY et al (2016) Trichoderma metabo-
lites as biological control agents against Phytophthora pathogens.
Biol Control 92:128–138. https://doi.org/10.1016/j.biocontrol
.2015.10.005
Jaroszewski JW, Stærk D, Boe Holm-Møller S et al (2005) Naravelia
zeyanica: occurrence of primary benzamides in flowering plants.
Nat Prod Res 19:291–294. https://doi.org/10.1080/1478641041
0001714641
Bai N, Wang S, Sun P et al (2018) Degradation of nonylphenol poly-
ethoxylates by functionalized Fe3O4 nanoparticle-immobilized
Sphingomonas sp. Y2. Sci Total Environ 615:462–468. https://
doi.org/10.1016/j.scitotenv.2017.09.290
Joe MM, Devaraj S, Benson A, Sa T (2016) Isolation of phosphate
solubilizing endophytic bacteria from Phyllanthus amarus Schum
& Thonn: evaluation of plant growth promotion and antioxidant
activity under salt stress. J Appl Res Med Aromat Plants 3:71–77.
https://doi.org/10.1016/j.jarmap.2016.02.003
Dai Z-C, Fu W, Wan L-Y et al (2016) Different growth promoting
effects of endophytic bacteria on invasive and native clonal plants.
Front Plant Sci 7:706. https://doi.org/10.3389/fpls.2016.00706
Doke N (1983) Generation of superoxide anion by potato tuber pro-
toplasts during the hypersensitive response to hyphal wall com-
ponents of Phytophthora infestans and specific inhibition of the
reaction by suppressors of hypersensitivity. Physiol Plant Pathol
23:359–367. https://doi.org/10.1016/0048-4059(83)90020-6
Egamberdieva D, Wirth SJ, Shurigin VV et al (2017) Endophytic bac-
teria improve plant growth, symbiotic performance of chickpea
(Cicer arietinum L.) and induce suppression of root rot caused by
Fusarium solani under salt stress. Front Microbiol 8:1–13. https
://doi.org/10.3389/fmicb.2017.01887
Kadowaki MAS, Müller-Santos M, Rego FGM et al (2011) Identi-
fication and characterization of PhbF: A DNA binding protein
with regulatory role in the PHB metabolism of Herbaspiril-
lum seropedicae SmR1. BMC Microbiol 11:230. https://doi.
org/10.1186/1471-2180-11-230
Kalaji HM, Jajoo A, Oukarroum A et al (2016) Chlorophyll a fluores-
cence as a tool to monitor physiological status of plants under
abiotic stress conditions. Acta Physiol Plant 38:102. https://doi.
org/10.1007/s11738-016-2113-y
Kanaly RA, Harayama S (2000) Biodegradation of high-molecular-
weight polycyclic aromatic hydrocarbons by bacteria. J Bacteriol
182:2059–2067
Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys
82:70–77. https://doi.org/10.1016/0003-9861(59)90090-6
Etesami H, Alikhani HA, Hosseini HM (2015) Indole-3-acetic acid
(IAA) production trait, a useful screening to select endophytic and
rhizosphere competent bacteria for rice growth promoting agents.
MethodsX 2:72–78. https://doi.org/10.1016/j.mex.2015.02.008
Fujiwara H, Soda S, Fujita M, Ike M (2016) Kinetics of bisphenol a
degradation by Sphingomonas paucimobilis FJ-4. J Biosci Bioeng
122:341–344. https://doi.org/10.1016/j.jbiosc.2016.02.015
Gao J, Wang Y, Wang CW, Lu BH (2014) First report of bacterial root
rot of ginseng caused by pseudomonas aeruginosa in China. Plant
Dis 98:1577. https://doi.org/10.1094/PDIS-03-14-0276-PDN
Ghavami N, Alikhani HA, Pourbabaee AA, Besharati H (2016) Study
the effects of siderophore-producing bacteria on zinc and phos-
phorous nutrition of canola and maize plants. Commun Soil
Sci Plant Anal 47:1517–1527. https://doi.org/10.1080/00103
624.2016.1194991
Kang S-M, Khan AL, Waqas M, You Y-H et al (2014) Plant growth-
promoting rhizobacteria reduce adverse effects of salinity and
osmotic stress by regulating phytohormones and antioxidants
in Cucumis sativus. J Plant Interact 9:673–682. https://doi.
org/10.1080/17429145.2014.894587
Kar M, Mishra D (1976) Catalase, peroxidase, and polyphenoloxidase
activities during rice leaf senescence. Plant Physiol 57:315–319
Kera Y, Abe K, Kasai D et al (2016) Draft genome sequences of Sphin-
gobium sp. strain TCM1 and Sphingomonas sp. strain TDK1,
haloalkyl phosphate flame retardant- and plasticizer-degrading
bacteria. Genome Announc 4:e00668–16. https://doi.org/10.1128/
genomeA.00668-16
Khan AL, Waqas M, Kang S-M et al (2014) Bacterial endophyte
Sphingomonas sp. LK11 produces gibberellins and IAA and pro-
motes tomato plant growth. J Microbiol 52:689–695. https://doi.
org/10.1007/s12275-014-4002-7
Gong B, Wu P, Huang Z et al (2016) Enhanced degradation of phenol
by Sphingomonas sp. GY2B with resistance towards subopti-
mal environment through adsorption on kaolinite. Chemosphere
148:388–394. https://doi.org/10.1016/j.chemosphere.2016.01.003
Gururani M, Mohanta T, Bae H (2015) Current understanding of the
interplay between phytohormones and photosynthesis under
environmental stress. Int J Mol Sci 16:19055–19085. https://doi.
org/10.3390/ijms160819055
Khan AL, Halo BA, Elyassi A et al (2016) Indole acetic acid and ACC
deaminase from endophytic bacteria improves the growth of Sola-
num lycopersicum. Electron J Biotechnol 21:58–64. https://doi.
org/10.1016/j.ejbt.2016.02.001
Knoth JL, Kim SH, Ettl GJ, Doty SL (2014) Biological nitrogen fixa-
tion and biomass accumulation within poplar clones as a result of
1 3

Archives of Microbiology
benzamides and their 2-alkoxy derivatives as possible antifungal
agents. Eur J Med Chem 39:867–872. https://doi.org/10.1016/j.
ejmech.2004.06.003
inoculations with diazotrophic endophyte consortia. New Phytol
201:599–609. https://doi.org/10.1111/nph.12536
Kumar SM, Kumar SR, Supriya S et al (2014) Isolation and characteri-
zation of siderophore producing antagonistic rhizobacteria against
Rhizoctonia solani. J Basic Microbiol 54:585–597. https://doi.
org/10.1002/jobm.201200564
Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in ani-
mal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–
358. https://doi.org/10.1016/0003-2697(79)90738-3
Oteino N, Lally RD, Kiwanuka S et al (2015) Plant growth promo-
tion induced by phosphate solubilizing endophytic Pseudomonas
isolates. Front Microbiol 6:745. https://doi.org/10.3389/fmicb
.2015.00745
Kumari R, Subudhi S, Suar M et al (2002) Cloning and characteriza-
tion of lin genes responsible for the degradation of Hexachloro-
cyclohexane isomers by Sphingomonas paucimobilis strain B90.
Appl Environ Microbiol 68:6021–6028. https://doi.org/10.1128/
AEM.68.12.6021-6028.2002
Ratcliff WC, Kadam SV, Denison RF (2008) Poly-3-hydroxybutyrate
(PHB) supports survival and reproduction in starving rhizo-
bia. FEMS Microbiol Ecol 65:391–399. https://doi.org/10.111
1/j.1574-6941.2008.00544.x
Kweon O, Kim SJ, Kim DW et al (2014) Pleiotropic and epistatic
behavior of a ring-hydroxylating oxygenase system in the poly-
cyclic aromatic hydrocarbon metabolic network from Mycobacte-
rium vanbaalenii PYR-1. J Bacteriol 196:3503–3515. https://doi.
org/10.1128/JB.01945-14
Ronca S, Frossard A, Guerrero LD et al (2015) Draft genome sequence
of Sphingomonas sp. strain Ant20, isolated from oil-contaminated
soil on Ross Island. Antarctica Genome Announc 3:e01309–
e01314. https://doi.org/10.1128/genomeA.01309-14
Ryan RP, Germaine K, Franks A et al (2008) Bacterial endophytes:
recent developments and applications. FEMS Microbiol Lett
278:1–9
Li D, Yan Y, Ping S et al (2010) Genome-wide investigation and
functional characterization of the β-ketoadipate pathway
in the nitrogen-fixing and root-associated bacterium Pseu-
domonas stutzeriA1501. BMC Microbiol 10:36. https://doi.
org/10.1186/1471-2180-10-36
Santoyo G, Moreno-Hagelsieb G, del Carmen Orozco-Mosqueda M,
Glick BR (2016) Plant growth-promoting bacterial endophytes.
Microbiol Res 183:92–99. https://doi.org/10.1016/j.micre
s.2015.11.008
Lichtenthaler H, Wellburn A (1983) Determinations of total carot-
enoids and chlorophylls a and b of leaf extracts in different sol-
vents. Biochem Soc Trans 11:591 LP–L592
Liu D, Jin X, Sun X et al (2016) Sphingomonas faucium sp. nov., iso-
lated from canyon soil. Int J Syst Evol Microbiol 66:2847–2852.
https://doi.org/10.1099/ijsem.0.001064
Stępniewska Z, Kuźniar A (2013) Endophytic microorganisms—prom-
ising applications in bioremediation of greenhouse gases. Appl
Microbiol Biotechnol 97:9589–9596. https://doi.org/10.1007/
s00253-013-5235-9
Ma T, Zhou Y, Li X et al (2016) Genome mining of astaxanthin bio-
synthetic genes from Sphingomonas sp. ATCC 55669 for heter-
ologous overproduction in Escherichia coli. Biotechnol J 11:228–
237. https://doi.org/10.1002/biot.201400827
Tabata M, Ohtsubo Y, Ohhata S et al (2013) Complete genome
sequence of the γ-hexachlorocyclohexane-degrading bacterium
Sphingomonas sp. Strain MM-1. Genome Announc 1:e00247–
e00213. https://doi.org/10.1128/genomeA.00247-13
Tamura K, Stecher G, Peterson D et al (2013) MEGA6: molecular evo-
lutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–
2729. https://doi.org/10.1093/molbev/mst197
MacLean AM, MacPherson G, Aneja P, Finan TM (2006) Characteri-
zation of the β-Ketoadipate Pathway in Sinorhizobium meliloti.
Appl Environ Microbiol 72:5403–5413. https://doi.org/10.1128/
AEM.00580-06
Manickam N, Bajaj A, Saini HS, Shanker R (2012) Surfactant mediated
enhanced biodegradation of hexachlorocyclohexane (HCH) iso-
mers by Sphingomonas sp. NM05. Biodegradation 23:673–682.
https://doi.org/10.1007/s10532-012-9543-z
Ullah A, Mushtaq H, Fahad S et al (2017) Plant growth promoting
potential of bacterial endophytes in novel association with Olea
ferruginea and Withania coagulans. Microbiology 86:119–127.
https://doi.org/10.1134/S0026261717010155
Mei C, Flinn B (2010) The use of beneficial microbial endophytes
for plant biomass and stress tolerance improvement. Recent Pat
Biotechnol 4:81–95
Zhang X, Liu X, Wang Q et al (2014) Diesel degradation potential of
endophytic bacteria isolated from Scirpus triqueter. Int Biode-
terior Biodegradation 87:99–105. https://doi.org/10.1016/j.ibiod
.2013.11.007
Miller TR, Delcher AL, Salzberg SL et al (2010) Genome sequence of
the dioxin-mineralizing bacterium Sphingomonas wittichii RW1.
J Bacteriol 192:6101–6102. https://doi.org/10.1128/JB.01030-10
Mohanta TK (2013) Plant metabolomics: missing link in next genera-
tion functional genomics era. J Appl Biol Biotechnol 1:1–10. https
://doi.org/10.7324/JABB.2013.1301
Zhu X, Ni X, Waigi MG et al (2016) Biodegradation of Mixed PAHs
by PAH-degrading endophytic bacteria. Int J Environ Res Public
Health 13:805. https://doi.org/10.3390/ijerph13080805
Mulla SI, Wang H, Sun Q et al (2016) Characterization of triclosan
metabolism in Sphingomonas sp. strain YL-JM2C. Sci Rep
6:21965
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Narayana B, Vijaya Raj KK, Ashalatha BV et al (2004) Synthe-
sis of some new 5-(2-substituted-1,3-thiazol-5-yl)-2-hydroxy
1 3