Screening of Microorganisms Capable of Biotransforming Certain Monoterpenes Using Substrate Toxicity Test

Abstract

Monoterpenes, such as Geraniol (G), Geranyl acetate (GA), Citral (CT), Limonene (LN), and Linalool (LL), are the most widely used phytochemicals in the aroma, food, and pharmaceutical industries. Here, we screened several bacteria and fungi to assess their potential to biotransform the selected monoterpenes (G, GA, CT, LN, and LL) through the substrate toxicity test. Three bacteria Pseudomonas fluorescens MTCC2421, Streptococcus mutans MTCC497, and Escherichia coli were found to be resistant to G, GA, and LN while two P. aeruginosa, and S. epidermidis MTTC 435 to GA and LN. In general, all fungal strains did not show resistance to any of the monoterpenes used, except Candida albicans and Fusarium oxysporum, which were slightly resistant to lower concentrations (0.05-0.1%) of GA. Interestingly, none of the bacteria and fungi showed any resistance to CT. The maximum concentrations of monoterpenes to which bacteria exhibited resistance ranged from 0.05-0.2%. The growth and biomass profiles of bacteria revealed that P. fluorescens and S. mutans grew well in the presence of monoterpenes GA and LN. Based on this, Pseudomonas fluorescens was capable of biotransforming GA and LN, while S. mutans only LN. The biotransformation of GA by P. fluorescens produced G and LL on the day 5th and 7th of the incubation. Hence, the study revealed the three potential bacteria, which may be useful in producing new aromatic derivatives from selected monoterpenes through biotransformation.

Full text

Citaon: Mial R, Srivastava G, Ganjewala D. Screening of Microorganisms Capable of Biotransforming Certain Monoterpenes
Using Substrate Toxicity Test. J Pure Appl Microbiol. Published online 28 February 2024. doi: 10.22207/JPAM.18.1.33
© The Author(s) 2024. Open Access. This arcle is distributed under the terms of the Creave Commons Aribuon 4.0 Internaonal License which
permits unrestricted use, sharing, distribuon, and reproducon in any medium, provided you give appropriate credit to the original author(s) and
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Mial et al | Arcle 8682
J Pure Appl Microbiol. 2024. doi: 10.22207/JPAM.18.1.33
Received: 08 May 2023 | Accepted: 09 January 2024
RESEARCH ARTICLE OPEN ACCESS
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P-ISSN: 0973-7510; E-ISSN: 2581-690X
*Correspondence: deepakganjawala73@yahoo.com
Screening of Microorganisms Capable of
Biotransforming Certain Monoterpenes Using
Substrate Toxicity Test
Ruchika Mial, Gauri Srivastava and Deepak Ganjewala*
Amity Instute of Biotechnology, Amity University Uar Pradesh, Sector 125, Noida, Uar Pradesh, India.
Abstract
Monoterpenes, such as Geraniol (G), Geranyl acetate (GA), Citral (CT), Limonene (LN), and Linalool (LL),
are the most widely used phytochemicals in the aroma, food, and pharmaceucal industries. Here, we
screened several bacteria and fungi to assess their potenal to biotransform the selected monoterpenes
(G, GA, CT, LN, and LL) through the substrate toxicity test. Three bacteria Pseudomonas uorescens
MTCC2421, Streptococcus mutans MTCC497, and Escherichia coli were found to be resistant to G, GA,
and LN while two P. aeruginosa, and S. epidermidis MTTC 435 to GA and LN. In general, all fungal strains
did not show resistance to any of the monoterpenes used, except Candida albicans and Fusarium
oxysporum, which were slightly resistant to lower concentraons (0.05-0.1%) of GA. Interesngly, none
of the bacteria and fungi showed any resistance to CT. The maximum concentraons of monoterpenes
to which bacteria exhibited resistance ranged from 0.05-0.2%. The growth and biomass proles of
bacteria revealed that P. uorescens and S. mutans grew well in the presence of monoterpenes GA
and LN. Based on this, Pseudomonas uorescens was capable of biotransforming GA and LN, while S.
mutans only LN. The biotransformaon of GA by P. uorescens produced G and LL on the day 5th and
7th of the incubaon. Hence, the study revealed the three potenal bacteria, which may be useful in
producing new aromac derivaves from selected monoterpenes through biotransformaon.
Keywords: Monoterpenes, Citral, Geraniol, Geranyl Acetate, Limonene, Biotransformaon, Substrate Toxicity Test

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INTRODUCTION
Monoterpenes are C10-containing
compounds that belong to the isoprenoids family
of secondary metabolites. They are the main
constituents of the essential oils of aromatic
plants. They impart a unique aroma to the
essential oils. Several monoterpenes such as
geraniol (G), geranyl acetate (GA), citral (CT),
limonene (LN), and linalool (LL) are highly popular
and widely used in fragrances, cosmecs, hygiene,
household products, food, and pharmaceucals.1-4
They exhibited a wide range of biological
acvies such as anbacterial, analgesic, an-
inammatory, ancancer, andiabec, anobesity,
and modulators of the gut microbiota.1,5,6 Citral
and linalool are also involved in the synthesis of
vitamins A, E, and ionones.7-9 At present, many
reports are being published on geraniol and citral,
demonstrang their potenal ancancer eects
and their scope in alternave cancer therapy.10
With the rapidly increasing importance
of monoterpenes in the producon of human
fragrances and tastes, demand for new
monoterpenes on the market will connue to
rise. At present, most of the avouring products
available in the market are produced by chemical
synthesis. Besides, many such products are also
isolated from plants by solvent extracon and
hydro-disllaon. However, chemical synthesis
has several demerits, such as the formaon of
undesirable chemical mixtures, inappropriate high
operang temperatures, and the side or adverse
eects of chemically synthesized products. In view
of this, consumers now prefer products, which
are produced by green synthesis to chemically
synthesized products. However, in plants, these
products are produced in a very limited quanty,
so plants may not be reliable sources for large-scale
extracon of such compounds. Therefore, microbial
biotransformation relying on microorganisms
and their biocatalysts has been proposed as an
alternave approach for the producon of novel
monoterpenoids. This has several advantages
over chemical processes. Recently, Mial et al.
studied the biotransformaon of monoterpenes by
microorganisms and plant cell and organ cultures.11
Considering the increasing and vastly varied
signicance of monoterpenes viz., geraniol (G),
geranyl acetate (GA), citral (CT), limonene (LN), and
linalool (LL) in the aroma industry and expanding
the field of microbial biotransformation, the
present study has been undertaken to screen
monoterpene resistant microorganisms from soil
samples, which may be used for the producon of
important monoterpenes through biotechnological
approaches. The monoterpene-resistant microbes
were screened using the substrate toxicity test.
MATERIALS AND METHODS
Chemicals
Authenc geraniol, geranyl acetate, citral,
limonene, and linalool were procured from Sigma-
Aldrich, India.
Microorganisms
Ten bacteria namely Escherichia
coli (MTCC901), Pseudomonas aeruginosa,
Pseudomonas uorescens, Pseudomonas puda,
Staphylococcus aureus (MTCC96), Streptococcus
mutans MTCC497, Staphylococcus epidermidis
MTTC 435, Shigella boydii MCC 2408, Acinetobacter
baumannii, Bacillus mycoides and the three fungi
Alternaria brushicicola, Fusarium oxysporum and
Candida albicans were obtained from the CSIR-
Instute of Microbial Technology, Chandigarh,
India. Bacteria were inoculated on nutrient agar
(NAM) and fungal cultures on potato dextrose
agar (PDA). Pure colonies were sub-cultured and
stored on slant agar at 4°C and 80% glycerol stocks
at -20°C.
Substrate-toxicity test
Substrate toxicity was performed to
screen monoterpene-resistant microorganisms
in accordance with previous methods.12,13 The
culture plates were prepared by displacing 30
ml sterilized NAM in pre-sterilized Petri dishes.
Each 1 ml (1.0 x 105 CFU/ml) inoculum is evenly
distributed to the agar medium with a sterile glass
rod. Wells were bored in agar plates using a sterile
cork borer (6 mm). To the wells, 25, 50, 75, and
100 µl of monoterpenes equal to concentraons
0.05-0.2% were added. Bacterial and fungal plates
were incubated separately at 37°C, 24 h, and 27°C,
48 h, respecvely. Simultaneously, posive and
negave control plates were also incubated. The

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plates were observed and the mean diameter of
the inhibion zone (mm) was measured. Each
experiment was performed in triplicate.
Microbial growth rates
The cultures were incubated in a rotary
shaker at 30°C and 275 rpm for seven days to
measure microbial growth. Bacterial growth rates
were measured in terms of absorbance at 660
nm. The biomass of fungal strains was ltered and
assessed by wet weight. Finally, the microbiological
growths were compared to a control without
monoterpenes.
Table 1. Monoterpene-resistant behavior of the bacterial strains
Bacterial Strains Concen. Zone of inhibion (mm)
(%) Geraniol Geranyl Citral Linalool Limonene
Acetate
Pseudomonas uroscens 0.05 NO NO 35 15 NO
MTCC 2421 0.1 NO NO 45 20 NO
0.15 NO NO 55 26 NO
0.2 NO NO 78 32 NO
P. aerginousa 0.05 15 No 30 13 NO
0.1 20 No 38 20 NO
0.15 28 No 45 26 NO
0.2 36 No 45 26 NO
Staphylococcus epidermidis 0.05 No 15 24 No 13
MTTC 435 0.1 No 21 33 No 20
0.15 No 29 42 No 25
0.2 No 35 48 No 29
Streptococcus mutans 0.05 No No 35 12 No
MTCC497 0.1 No No 40 16 No
0.15 No No 40 20 No
0.2 No No 50 20 No
E. coli 0.05 11 No 40 No No
0.1 20 No 60 No No
0.15 32 No 90 No No
0.2 32 No 90 No No
Shigella boydii MTCC2408 0.05 11 No 18 18 25
0.1 15 10 25 28 25
0.15 20 16 30 40 25
0.2 25 25 30 40 25
Acinetobacter baumanaii 0.05 10 No 15 24 No
0.1 18 15 25 30 1
0.15 20 20 25 30 15
0.2 28 20 30 35 15
S. aureus 0.05 23 No 20 20 20
0.1 15 No 20 20 20
0.15 15 15 20 24 25
0.2 20 15 30 24 25
P. puda 0.05 14 13 30 20 18
0.1 20 13 40 20 24
0.15 27 19 50 24 30
0.2 35 26 60 24 30
Mycoid 0.05 14 12 20 20 20
0.1 26 25 20 20 20
0.15 35 25 20 24 25
0.2 48 25 20 24 25

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Biotransformaon assay
Biotransformaon of GA by P. uorescens
was performed in 150-mL Erlen Mayer flasks
containing 50 ml nutrient broth medium (yeast
extract 2 gL−1, beef extract gL−1, peptone 5 gL−1,
sodium chloride 5 gL−1, pH 7). An inoculum of P.
uorescens and GA (each 25μl) was added to the
nutrient medium and the bacteria were allowed
to grow on an orbital shaker at 150 rpm and 37°C.
Samples (5 ml) were asepcally taken from the
cultures at regular intervals (24 h) for 7-10 days.
Two controls were used: a media-only (without the
inoculums and substrate), and bacterial control
without substrate.
Biotransformaon products of GA
The biotransformation products from
the samples were extracted aer removing the
bacterial cells by centrifugaon. The supernatant
was extracted thrice with 25 ml of diethyl
ether. Thus, the pooled extract was washed
three mes with dislled water (10 ml), dried
over anhydrous sodium sulfate, and filtered
through Whatman paper No. 1. The sample was
evaporated to dryness and subjected to thin-layer
chromatographic separaon (TLC).14 Samples and
standards were loaded directly onto silica gel-G
plates. The plates were developed in a solvent
system consisng of toluene: ethyl acetate (96:4
v/v) at 4°C. The plates were then removed and
dried at room temperature. Spots were visualized
by exposing plates to iodine vapour. Idencaon
of spots was done by comparing relave frontal
(Rf) values of the standards used. Analysis of the
biotransformaon products was also performed by
gas chromatography-mass spectrometry (GC-MS).
Stascal analysis
The mean and standard deviation of
minimum inhibion zone (MIZ) diameter (mm)
were calculated based on percent zone reducon
in comparison to the control plate.
RESULTS
Biotransformaon potenal of microbes
The results of the substrate toxicity
tests are presented in Tables 1 and 2. Five of the
ten bacterial strains P. uorescens MTCC2421, P.
aeruginosa, S. mutans MTCC497, S. epidermidis
MTTC435P, and E. coli were found to be resistant
while the remaining ve Shigella boydii MTCC2408,
P. puda, A. baumanaii, Mycoid, and S. aureus
highly sensive to all monoterpenes used. Four
bacteria P. uorescens MTCC2421, P. aeruginosa,
S. mutans MTCC497, and E. coli, showed resistance
to GA and LN at all concentraons 0.05-0.2%.
Three bacteria including P. uorescens MTCC2421
and E. coli and S. epidermidis MTTC435P showed
resistance to both G and LL, whereas the other
three P. aeruginosa, S. mutans MTCC497, and E.
coli were found sensive to G. Among all, only
S. epidermidis MTTC435P was suscepble to GA
and LN. Three other P. uorescens MTCC2421,
Table 2. Monoterpene-resistant behavior of the fungal strains
Fungal Strains Concen. Zone of inhibion (mm)
(%) Geraniol Geranyl Citral Linalool Limonene
Acetate
Candida albanicas 0.05 14 No 40 No 18
0.1 20 No 50 15 25
0.15 24 15 65 20 32
0.2 30 15 75 20 38
Alternaria brushicicola 0.05 25 45 65 65 65
0.1 33 54 70 76 78
0.15 45 69 90 90 90
0.2 60 80 90 90 90
Fusarium oxysporum 0.05 15 No 35 30 25
0.1 20 No 45 42 25
0.15 20 14 60 50 36
0.2 20 14 80 80 42

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Figure 2. Eect of monoterpenes (0.05%) on the growth of P. aeruginosa
Figure 1. Eect of monoterpenes (0.05%) on the growth of P. uoresens
P. aeruginosa, S. mutans MTCC497 were highly
sensive to linalool. Interesngly, all bacterial
strains were suscepble to CT at all concentraons
from 0.05-0.2% but three of them namely P.
uorescens MTCC2421, E. coli, and P. puda were
highly susceptible with the zone of inhibition
values of 30-90 mm (Table 1). The toxicity assay
revealed that all the fungal strains were very
sensitive to all monoterpenes used; however,
C. albicans and F. oxysporum showed little
resistance to GA (0.05%) (Table 2).
Analysis of biomass proles
Biomass of P. uorescens, P. aeruginosa,
and S. mutans accumulated in the media in the
presence of G, GA, and LN was measured recording
the absorbance at 660 nm and compared with the
control (Figure 1-3). For P. uorescens MTCC242,

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Figure 3. Eect of monoterpenes (0.05%) on the growth of S. mutans
Figure 4. A thin-layer chromatogram of the
biotransformaon products of geranyl acetate. Lines
1-7 represent the incubaon period from 1-7 days
the biomass accumulated was highest at ~100%
during 1-4 days in the presence of GA and LN,
which slightly decreased by 4% in GA but increased
by 13% in LN on day 7 as compared to the control
(Table 3). However, the biomass decreased
signicantly from 35 to 56% in the presence of
G during the incubaon period of 1-7 days. We
did not observe any biomass accumulaon for P.
aeruginosa during the incubaon period in the
presence of G (Table 2). Overall, the biomass of
bacteria declined signicantly by 50-100% in the
presence of GA and decreased by a comparavely
very low margin of 10% in the presence of LN. In
the case of S. mutans, the biomass was highest
at ~118% in the presence of GA and LN on day 7
compared to the control. The biomass, however,
decreased from 10 to 21% in the presence of G and
LN from 1-4 days of incubaon and again increased
by 100% and 118% at day 7 (Table 3). Thus, these
results revealed that P. uorescens MTCC242 and
S. mutans are suitable for the biotransformaon
of GA and LN and P. aeruginosa for LN.
Biotransformaon of GA
The main product of the biotransformaon
of GA by P. uorescens was G (Figure 4). Geraniol
was rst detected on the day 5th of incubaon,
which was transformed into LL on the 7th day
(Figure 4). In addion, some other products were
produced, which could not be resolved on TLC,
most likely they were hydrocarbons. The rate of
biotransformaon of GA varied with the incubaon
me. On the day 5th, the biotransformaon of GA
using P. uorescens produced 50% geraniol. The
presence of GA and G was further conrmed by
GC-MS (Figure 5).

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DISCUSSION
Microbial biotransformation is a fast-
growing alternave method of chemical synthesis
for the production of many human products
such as avors, fragrances, food addives, and
more. This method relies on microbes (bacteria,
fungi, and yeast) and their enzymes, which are
capable of transforming selected compounds into
desirable products with mulple benets. Since
the availability of potenal microbes is the rst
essenal requirement for any biotransformaon,
in the present study, we screened a few bacteria
that can transform the monoterpenes G, GA, LL,
and LN through a substrate toxicity assay.
Results of the toxicity assay revealed that
microbes had varying degrees of tolerance to the
selected monoterpenes used at concentraons
Table 3. Biomass (%) of monoterpene-resistant bacteria
Treatment Days Biomass accumulaon (%)
P. uroscens P. aeruginosa S. mutans
MTCC 2421 MTCC497
Control 1st 100 100 100
4
th 88 94 95
7
th 82 94 79
Geraniol 1st 64 00 79
4
th 53 00 83
7
th 44 00 100
Geranyl acetate 1st 100 49 100
4
th 100 28 99
7
th 96 00 118
Limonene 1st 100 89 89
4
th 87 89 87
7
th 113 100% 118
Figure 5. The GC-MS shows the biotransformaon products of geranyl acetate on the day 1 (A) and day 5 (B) of
incubaon

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from 0.05-0.2% (Tables 1 and 2). It was found
that P. uorescens and S. epidermidis MTTC 43
eecvely tolerated G (0.2%), while others were
unable to tolerate >0.05% G. The toxicity test
indicated that P. aeruginosa, P. uorescens, S.
mutans MTCC497, and E. coli MTCC901 tolerated
0.05-0.2% GA while S. boydii MCC2408, A.
baumanaii, and S. aureus could survive only in
the presence of 0.05% GA. Two bacterial strains S.
epidermidis MTTC 435 and E. coli MTCC901 grew
well in the presence of 0.05-0.2% LL, whereas the
other four P. aeruginosa, P. uorescens, S. mutans
MTCC497, and E. coli MTCC901 in 0.05-0.2% LN.
Fungal strains C. albicans and F. oxysporum were
more sensive and could survive only in 0.05-0.1%
GA (Table 2). These results revealed a correlaon
between microbial growth and monoterpene
concentraons. In general, microbes exhibited
resistance to varying concentrations (0.05 to
0.2%) of monoterpenes. Hence, any of these
concentrations may be used to perform the
microbial biotransformaon of the monoterpenes.
Several previous studies have also suggested a
concentraon range of monoterpenes from 0.1
to 0.2% most suitable for their biotransformaon
by Pseudomonas, Saccharomyces spp. and
Penicillium, Aspergillus spp.13,15-17
Although simple microbial resistance to
monoterpenes with added carbon sources does
not guarantee high biotransformaon acvity, it is
an essenal property of a biotransformaon agent.
Therefore, we performed initial physiological
studies to characterize microbial growth behaviour
in the presence of monoterpenes. Two bacteria
P. uorescens and S. mutans showed the best
growth proles in the presence of GA. The biomass
content of these bacteria was almost equal to the
control throughout the incubaon of 1-7 days.
However, the biomass of P. aeruginosa signicantly
declined by 51-100%. These results suggest the
rapid consumption of GA in P. fluorescens, S.
mutans, and P. aeruginosa, which are most likely
to have substrate-degrading metabolic pathways.15
The biomass is directly proporonal to the growth
rates, therefore, the higher the biomass the higher
will be the growth. Here, the maximum microbial
growth was recorded within the rst two days of
incubaon compared to the control. Fungal growth
was reduced only at the lower concentraons
(0.05%) of GA on day 1 of incubaon (Table 2).
Thus, the substrate toxicity test and
biomass accumulation profiles suggest that P.
uorescens and S. mutans MTCC497, P. aeruginosa
have the potenal for biotransformaon of GA
and LN. Previous studies have reported that
P. fluorescens and P. putida biotransformed
limonene into limonene-1,2-oxide and perillyl
alcohol.18,19 In contrast, in the present study, P.
puda was found to be sensive to limonene.
Besides the biotransformaon of GA and LN, the
biotransformaon of geraniol can be carried out
by S. mutans MTCC497 and P. uorescens. Earlier,
we reported an enzyme, geranyl acetate esterase
(GAE) from lemongrass leaves that catalyzes
the biotransformaon of GA into G.20 However,
in the literature, no report is available on the
biotransformaon of GA by microbial enzymes.
Here, we carried out the biotransformaon of
GA by P. uorescens producing G, LL, and other
products (Figures 4 and 5). This action of P.
fluorescens can be attributed to homologous
esterase and synthase enzymes. In accordance
with a previous study, opmizaon of several
parameters like the catalyst, reacon medium,
stirring rate, molar ratio, and temperature is
being carried out to improve the eciency of the
microbial biotransformaon system.21 Certainly,
the outcomes of this study may be used to carry
out the biotransformation of geranyl acetate,
geraniol, and other monoterpenes to produce
newer commercial aromac derivaves.
CONCLUSION
The present study revealed three
potenal bacteria P. uorescens, S. mutans, and
P. aeruginosa with an ability to biotransform
GA, G, and LN. However, none of the fungi was
found capable of biotransforming the selected
monoterpenes. The biotransformation with
various monoterpenes can be carried out ulizing
parcular bacteria in order to choose the nest
strains benecial for industrial applicaons. Thus,
the current work underlines the importance of the
screening of microorganisms as the rst step in the
biotransformaon processes.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Ashok
Kumar Chauhan, Founder President and Mr. Atul

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Chauhan, Chancellor of Amity University, Uar
Pradesh, Noida, India, for providing the necessary
facilies and support.
CONFLICT OF INTEREST
The authors declare that there is no
conict of interest.
AUTHORS' CONTRIBUTION
All authors listed have made a substanal,
direct and intellectual contribuon to the work,
and approved it for publicaon.
FUNDING
None.
DATA AVAILABILITY
All datasets generated or analyzed during
this study are included in the manuscript.
ETHICS STATEMENT
Not applicable.
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