Applied Nanoscience

Silver nanoparticles produced via a green synthesis using the rhamnolipid as a

reducing agent and stabilizer
--Manuscript Draft--

Manuscript Number: APNA-D-22-00377R1

Full Title: Silver nanoparticles produced via a green synthesis using the rhamnolipid as a
reducing agent and stabilizer

Article Type: Original Article

Abstract: Silver nanoparticles (AgNPs) stabilized by rhamnolipid (RL), which was separated from
the supernatant of the genus Pseudomonas sp. PS-17, were synthesized. Using TEM
it was found that the mean size of obtained AgNPs does not depend on the initial
concentration of Ag + but is increased with the decrease of the concentration of RL.
During XRD-phase analysis it was determined, that the Ag (space group Fm -3 m ,
Cu-structure type) is a main phase which was identified. The kinetics of the formation
of AgNPs was studied in details using the UV-vis spectroscopy. It was observed that in
the all cases the kinetic curves are sigmoidal shape and are characterized by well-
notable induction period that permits to assume the homogeneous nucleation of
AgNPs and their autocatalytic growth. Experimental kinetic curves were fitted using
different types of Finke-Watzky schemes of continuous nucleation and fast
autocatalytic growth of particles and the observable rate constants of nucleation and
growth were estimated. Based on the established regularities of “green” synthesis of
AgNPs stabilized by RL, the method of obtaining of their colloidal solutions was
optimized. A laboratory model of a flow tubular reactor for the synthesis of colloidal
solutions of AgNPs has been created.

Response to Reviewers: Dear Editors,

Presented manuscript is submitted to the Special Issue of the journal "Applied

Nanoscience" devoted to the International conference "Nanotechnology and
nanomaterials" (NANO-2021). Due to the conference was proceeded in combined
mode (offline and online), our own poster was placed on the web-site of the
Conference. And it is logical that some separate results (namely, the motivation of the
investigations and short result discussion) were presented on our own poster. Above
mentioned poster is not the publication, but only the partial presentation of our
obtained results in the work of the Conference. However, in accordance with Editors
recommendations, we have supplemented the Acknowledgement as follow:
“Some separated (the motivation of the investigations and short result discussion)
results obtained by the authors were partially presented as a poster on the
International conference "Nanotechnology and nanomaterials" (NANO-2021) (August
25-27, 2021, Lviv, Ukraine).”
The correction has been marked by yellow.

With kind regards,

Dr. Liliya Bazylyak

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SILVER NANOPARTICLES PRODUCED VIA A GREEN SYNTHESIS

USING THE RHAMNOLIPID AS A REDUCING AGENT AND STABILIZER
1
2
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4 Bazylyak L. I.1*, Kytsya A. R.1,3, Lyutyy P. Y.2,3, Korets’ka N. I.1, Pilyuk Ya. V.1, Kuntyi O. I.2
5
6
1
7 Department of Physical Chemistry of Fossil Fuels of the Institute of Physical–Organic Chemistry and Coal
8
9 Chemistry named after L. M. Lytvynenko of the National Academy of Sciences of Ukraine.
10 3а Naukova Str., Lviv, 79060, Ukraine;
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12 2
Lviv Polytechnic National University, 12 Bandery Str., 79013, Lviv, Ukraine;
13
14 3
15 Karpenko Physico–Mechanical Institute of the National Academy of Sciences of Ukraine
16 5 Naukova Str., Lviv, 79060, Ukraine
17
18 *
19 Corresponding author: Liliya Bazylyak, e-mail: bazyljak.L.I@nas.gov.ua
20
21 Abstract.
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23 Silver nanoparticles (AgNPs) stabilized by rhamnolipid (RL), which was separated from the supernatant
24
25 of the genus Pseudomonas sp. PS-17, were synthesized. Using TEM it was found that the mean size of
26 obtained AgNPs does not depend on the initial concentration of Ag+ but is increased with the decrease of the
27
28 concentration of RL. During XRD-phase analysis it was determined, that the Ag (space group Fm-3m, Cu-
29
30 structure type) is a main phase which was identified. The kinetics of the formation of AgNPs was studied in
31 details using the UV-vis spectroscopy. It was observed that in the all cases the kinetic curves are sigmoidal
32
33 shape and are characterized by well-notable induction period that permits to assume the homogeneous
34
35 nucleation of AgNPs and their autocatalytic growth. Experimental kinetic curves were fitted using different
36 types of Finke-Watzky schemes of continuous nucleation and fast autocatalytic growth of particles and the
37
38 observable rate constants of nucleation and growth were estimated. Based on the established regularities of
39
40 “green” synthesis of AgNPs stabilized by RL, the method of obtaining of their colloidal solutions was
41 optimized. A laboratory model of a flow tubular reactor for the synthesis of colloidal solutions of AgNPs has
42
43 been created.
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45 Keywords. Green synthesis, silver nanoparticles, rhamnolipid, kinetics, flow tubular reactor.
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 1. Introduction.
Silver nanoparticles (AgNPs) have great potential in a broad range of applications as antimicrobial
1
2 agents (Kuntyi et al. 2021), biomedical device coatings (Grytsenko et al. 2018), drug-delivery carriers,
3
4 imaging probes, diagnostic and optoelectronic platforms (Lee and Jun 2019), since they have discrete
5 physical, chemical and optical properties and biochemical functionality. Silver nanoparticles have been
6
7 investigated extensively due to their superior physical, chemical, and biological characteristics, and their
8
9 superiority stems mainly from the size, shape, composition, crystallinity and the structure of AgNPs
10 compared to their bulk forms (Syafiuddin et al. 2017; Kumar et al. 2008; Desireddy et al. 2013; Sun et al.
11
12 2002; Atwater et al. 2010; Koval’chuk et al. 2010). Recently in the literature, there have been reports of the
13
14 AgNPs obtaining in different biological solutions via the so-called “green methods”, for example, using plant
15 extract (Krishnaraj et al. 2010; Vilchis-Nestor et al. 2008; Chandran et al. 2006; Amooaghaie et al. 2015; Bar
16
17 et al. 2009; Some et al. 2018; Ahmad et al. 2019; Mousavi et al. 2018; Srikar et al. 2016) and supernatants
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19 obtained from bacterial (Płaza et al. 2016; Gurunathan et al. 2009; Shahverdi et al. 2007; Kalishwaralal et al.
20 2008; Nanda et al. 2009; Kuntyi et al. 2020) or fungus cultures (Vigneshwaran et al. 2007; Bhainsa et al.
21
22 2006; Kapoor et al. 2021). In this green chemistry approach, several bacteria, including Pseudomonas
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24 stutzeri AG259 (Klaus et al. 1999), Lactobacillus (Nair et al. 2002), Bacillus licheniformis (Kalimuthu et al.
25 2008), Escherichia coli (Gurunathan et al. 2009), Brevibacterium casei (Kalishwaralal et al. 2020), plant
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27 extracts such as Allophylus cobbe (Gurunathan et al. 2014), Artemisia princeps (Gurunathan et al. 2015),
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29 Typha angustifolia (Gurunathan et al. 2015) were used. In addition to these, several biomolecules, such as
30 biopolymers (Leung et al. 2010), starch (Kumar et al. 2014), fibrinolytic enzyme (Deepak et al. 2011), and
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32 aminoacids (Shankar and Rhim 2015) were used. Biological entities could act simultaneously as reducing
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34 and stabilizing agents. Increasing attention to the biological synthesis of AgNPs can be attributed to
35 environmental and economic reasons. Green synthesis of AgNPs with naturally occurring reducing agents
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37 could be a promising method to replace more complex physiochemical syntheses since the green synthesis is
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39 free from toxic chemicals and hazardous byproducts and instead involves natural capping agents for the
40 stabilization of AgNPs. Use of biological agents seems to provide controlled particle size and shape, which is
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42 an important factor for various biomedical applications (Gurunathan et al. 2014). The other advantages of
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44 biological agents are the availability of a vast array of biological resources, a decreased time requirement,
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high density, stability, and the ready solubility of prepared nanoparticles in water. Biogenic surfactants are
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47 one of promising agents for the development of nanocomposites, they are ecologically safe alternative to
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49 toxic chemicals. The most known biosurfactants are rhamnolipids, which represent by themselves the low
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molecular weight secondary metabolites of bacteria genera Pseudomonas.
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52 In previous article (Bazylak et al. 2022) we reported about high antimicrobial activity of AgNPs
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54 stabilized by RL against some test microorganisms such as Escherichia coli, Staphylococcus aureus,
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Pseudomonas aeruginosa, Mycrococcus luteus (human pathogenes) and Agrobacterium tumefaciens and
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57 Xanthomonas campestris (phytopathogenes). It was established, that the values of minimum bactericidal
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59 concentration (MBC) of AgNPs for the A. tumefaciens, X. campestris and M. luteus are very low and are
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61 equal to 1.5 g/mL. Therefore, the low values of MBC compared to the low cost of obtained “AgNPs-RL”
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 solutions make them attractive in terms of development of antimicrobial drugs with a broad spectrum of
action. That is why the main aim of presented work was to synthesize the AgNPs in the presence of
1
2 rhamnolipid, to investigate in detail the kinetics and mechanism of the AgNPs formation via the reduction of
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4 Ag+ by RL as well as to investigate an influence of experimental conditions on the properties of obtained
5 nanoparticles in order to design the method of continuous synthesis of “AgNPs-RL” solutions.
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2. Experimental Details
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9 2.1. Synthesis of AgNPs
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11 The rhamnolipid of natural origin (RL), which represents by itself the product of microbial synthesis
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13 of the strain Pseudomonas sp. PS-17 was used as the reducing agent and stabilizer for the “green” synthesis
14 of AgNPs. RL was separated accordingly to described earlier technique (Pokynbroda et al. 2019; Yerokhin et
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16 al. 2006; Karpenko et al. 2009) and was provided for the investigations by the scientific group led by
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18 professor Olena Karpenko (Department of Physical Chemistry of Fossil Fuels of the Institute of Physical-
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Organic Chemistry and Coal Chemistry named after L. M. Lytvynenko of the National Academy of Sciences
20
21 of Ukraine).
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23 In accordance with the typical synthesis 95 mL of solution of RL was added into two-necked flask
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equipped by magnetic stirrer and Dimroth-type condenser and placed into the thermostat. pH of the RL
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26 solution was regulated by NaOH (titrant, Kharkovreachem, Ukraine) and was controlled using the Adwa
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28 AD1000 pH/mV & Temperature meter (Adwa Instruments, Hungary). After the reaching of the operative
29
temperature, 5 mL of AgNO3 (Aldrich, >99.0 %) solution of a given concentration was added into the flask.
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31 Kinetics of the reaction was monitored via the change of UV–vis spectra of the working solution
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33 applying the UVmini-1240 spectrophotometer (Shimadzu, Japan) with the use of 1 cm cuvette at wavelength
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35
range from 300 to 1100 nm. The COPASI Software (Hoops et al. 2006) was used for the evaluation of the
36 observable rate constants of the process.
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38 The completeness of the reaction conversion was tested using the hydrazine test: after the finishing of
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40 the kinetic measurements the 0.05 mL of hydrazine hydrate (N2H4H2O, Systema Optimum, Ukraine,
41 >98.0 %) was added to 5 mL of the solution, and UV-vis spectrum of the sample was recorded. It should be
42
43 noted, that in the all of the investigated experimental conditions any changes of the spectra before and after
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45 of hydrazine addition were not observed. This fact proves the near 100 % degree of reduction of Ag+ to Ag0.
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47 2.2. Characterisation of AgNPs
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49
Obtained AgNPs were characterized using the transmission electron microscopy (TEM) and X-ray
50 diffraction analysis (XRD). TEM images of obtained AgNPs were recorded using the JEM-I230 (JEOL,
51
52 Japan). The size of AgNPs were evaluated using an AxioVision V 4.8.2.0 (Carl Zeiss Microscopy) software.
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54 The samples for TEM investigations were prepared by application of micro-drops (~0.5 μL) of silver sol on
55 the carbon grid with the subsequent drying under the ambient conditions.
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57 The samples for the XRD measurements were prepared by the concentrating of the AgNPs solution at
58
59 80 C with the subsequent precipitation of AgNPs by ethanol (96 %), decantation of the precipitate and
60 following drying under the ambient conditions.
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 X-ray powder diffraction data was obtained with a use of the X-ray diffractometer (Aeris-Malvern
Panalytical) with Cu-Kα radiation operated at a voltage of 40 kV, a current of 15 mA and setup, beam knife
1
2 high, start angle 25 (2θ), end angle 100, step size 0.0217.
3
4 XRD-phase analysis was performed using the Highscore software and referenced with the
5 International Center for Diffraction Data (ICDD). The all procedures including the indexing, structure
6
7 solutions and the refinement of profile and structural parameters were performed with the WinCSD
8
9 (Akselrud and Grin 2014) program package.
10
11 2.3. The scheme of laboratory plug flow reactor for the AgNPs synthesis
12
13 The constructed laboratory setup for the synthesis of AgNPs consisted of the thermostated serpentine
14 reactor (1) connected to the peristaltic pump (2) and circulation thermostat (3) (Fig. 1). Inner pipe diameter
15
16 was 4 mm and the pipe length was 800 mm; the total volume of a reactor was 10 mL. The working solution
17
18 (4) (RL + AgNO3) was fed into the reactor with the given rate, providing the complete reduction of Ag+ to
19 Ag0.
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42 Fig. 1 The scheme of laboratory setup for the continuous synthesis of AgNPs.
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3. Results and discussion
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46 3.1. Kinetics of “green” synthesis of AgNPs
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48 An influence of the synthesis conditions on the kinetics of Ag+ reduction by RL was studied in details
49
50 in order to optimize the method of obtaining of the AgNPs solutions. The kinetics has been studied using the
51
UV-vis spectroscopy via the change of the maximum of absorbance band of the surface plasmon resonance
52
53 (SPR) of AgNPs (Sandoe et al. 2019; Kytsya et al. 2019).
54
55
56 3.1.1. An influence of pH on the kinetic parameters of AgNPs formation.
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58
An influence of pH on the kinetics of AgNPs formation was studied at 70 0C (Fig. 2). The initial
59 concentrations of Ag+ and RL were 210–4 mol/L and 4 g/L respectively.
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62 4
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 1
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22 Fig. 2. The evolution of the spectra of working solution during the synthesis of AgNPs at pH=8.
23 Insertion is the changing of the maximum of SPR during the synthesis.
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25
26 It was found that the spectra of the solutions are characterized by a clear absorption maximum which
27
28 may indicate the spherical shape of the obtained AgNPs. At the same time, during the synthesis a shift of the
29 absorption maximum in the short-wavelength region was observed (Fig. 2, insertion). This phenomenon can
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31 be explained as follows. The position of SPR band is significantly influenced by the value of the dielectric
32
33 constant of the medium (Slistan-Grijalva et al.2005; Kytsya et al. 2014), and in the case of surfactants that
34 are adsorbed on the particle surface – by the interaction between surfactant molecules and surface atoms of
35
36 the growing silver particle. With increasing of the particle size during the synthesis, the ratio of surface
37
38 atoms that interact with the surfactant molecules is decreased, which, accordingly, affects the position of the
39 SPR band of the AgNPs solution. Since the ratio of the values of the current and the final optical density of
40
41 the solution is proportional to the conversion of the reaction a = O.D.t/O.D.max (Sandoe et al. 2019), the
42
43 obtained spectra were used for the plotting of the kinetic curves of the processes (Fig. 3). As it can be seen
44
from Fig. 3(a), the kinetic curves of the AgNPs formation are sigmoidal which indicates on the autocatalytic
45
46 nature of the process. From the obtained kinetic curves the time of induction period (tind) and the maximal
47
48 rate of the process (da/dt) were calculated (Table 1).
49
Kinetic curves (Fig. 3, a) indicate a significant effect of pH of the reaction mixture on the rate of the
50
51 formation of AgNPs. However, the rate of nucleation of AgNPs (1/tind) is proportional to the pH of the
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53 reaction medium (Fig. 3, b). At the same time, the dependence of the rate AgNPs growth on pH is
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55
nonmonotonic. Thus, based on the obtained data, we can assume that the optimal pH for the synthesis of
56 AgNPs stabilized by RL is pH = 10. Therefore, the all subsequent studies were performed at pH=10.
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 1
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14 Fig. 3. The kinetic curves of AgNPs formation at different pH (a) and the dependencies of the rates of
15
16 nucleation and growth of AgNPs on pH (b).
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18
19
3.1.2. An influence of initial concentrations of RL and AgNO3 on the kinetics of the AgNPs formation.
20 The dependence of the kinetics of AgNPs formation on the initial concentrations of RL and AgNO 3
21
22 were studied at 70 0C and pH=10.
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Fig. 4. The kinetic curves of AgNPs formation at different initial concentrations of RL (a) and AgNO3 (b).
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43 It was found that in the all of cases the nature of the evolution of absorption spectra during the
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45 synthesis of AgNPs is similar to the described above, namely in the all cases an increase of the absorption
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47 intensity over the time and a shift of the SPR band in the shortwave region were observed. Based on the
48 obtained spectra the kinetic curves of AgNPs were plotted (Fig. 4) and the kinetic parameters of the process
49
50 were determined (Table 1).
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14 Fig. 5. The dependencies of the rates of nucleation and growth of AgNPs on the initial concentrations
15 of RL (a) and AgNO3 (b).
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18 It was found that both the nucleation rate and the growth rate of AgNPs linearly depend on the initial
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20 concentration of rhamnolipid (RL) (Fig. 5, a). Thus, due to the fact that the concentration of rhamnolipid in
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the reaction mixture is much higher compared to the concentration of Ag+, we can assume the first order of
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23 the processes of nucleation and growth of AgNPs in respect to RL. At the same time, the dependencies of the
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25 nucleation rates and growth of AgNPs on the initial concentration of silver ions are close to linear too (Fig. 5, b).
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However, since the concentration of Ag+ is changed during the synthesis, as well as due to the autocatalytic
28 nature of the process, such dependences require an additional analysis (vide infra, Section 3.3).
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31 3.1.3. Activation parameters of the AgNPs formation.
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33 In order to establish the activation parameters of the silver ion reduction with rhamnolipid, the effect
34 of temperature on the kinetics of AgNPs formation was studied ynder the following synthesis conditions:
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36 [AgNO3]0 = 210–4 mol/L, RL = 4 g/L, pH = 10.
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54 Fig. 6. The kinetic curves of AgNPs formation at different temperatures (a) and the dependencies of
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56 the rates of nucleation and growth of AgNPs in Arrhenius coordinates (b).
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58 It was found that at 50 0C the spectra of the working solution does not changed during 250 min (Fig.6, a),
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60 i.e. the rate of the reduction of silver ions by rhamnolipid at this temperature is extremely slow. Based on the
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 values of the kinetic parameters of the process (Table 1), the activation energies of nucleation and growth of
AgNPs were calculated (Fig. 6, b), which are equal to 135 ± 35 and 140 ± 40 kJ/mol, respectively.
1
2
3 Table 1. Dependence of the kinetic parameters of the AgNPs formation on reaction conditions
4
5 рН [Ag+]0104, mol/L C(RL), g/L t, C tind, min da/dt, min–1
6
7 8 45 0.008
8
9 9 0.2 4 70 7 0.054
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11 11 3 0.09
12 0.2 5.0 0.08
13
14 10 0.5 4 70 3.3 0.13
15
16 1 1.8 0.25
17
18 1 9 0.04
19 10 0.2 70
2 7 0.06
20
21 50  0
22
23 10 0.2 4 60 40 0.01
24
25 80 2.5 0.14
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27
28 High and almost identical values of activation energies of the nucleation and the growth of silver
29 nanoparticles exclude the diffusion nature of the process of nucleation of a new phase as well as with a high
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31 probability indicate the same nature of the chemical processes that occur during the nucleation and growth of
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33 AgNPs.
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35
3.2. Characterization of AgNPs stabilized by RL
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37
38 Obtained AgNPs were characterized using the XRD and TEM methods.
39
40 During XRD-phase analysis of obtained sample (Fig. 7) the Ag (space group Fm-3m, Cu – structure
41 type) as a main phase was identified and the unknown small peaks in the 2 range 25–300, which don’t
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43 correspond to silver as well as to silver oxides were observed. The assumption that these peaks belong to
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45 remains of RL was made. To confirmed or disprove this, the XRD scan of RL (Fig. 7) was performed and
46 good match was found. Graphical representation of the Rietveld refinements of the powder data of the
47
48 sample is shown on Fig. S-1 (Supplemental Information). Refined lattice cell parameter for Ag is equal to a
49
50 = 4.050(1) Å.
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20 Fig. 7. XRD-pattern of AgNPs stabilized by RL and the scan of RL.
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22 Using TEM it was found that the obtained AgNPs are polydisperse and mostly irregular in shape
23
24 (Fig.8, a).
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Fig. 8. TEM-image and histogram of particle size distribution of AgNPs obtained at 70 0C and initial
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47 concentrations of RL and AgNO3 equal to 4 g/L and 110–3 mol/L respectively.
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49 However, a detailed analysis of TEM-images obtained at a high magnification (~100,000) revealed
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51 that large nanoparticles (10–40 nm) are surrounded by a “cloud” of small (1–4 nm) particles (Fig. 8, a,
52
53 insertion). A similar pattern is observed for the all studied samples and can be caused by the method of
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preparation of samples for TEM, namely: for TEM investigations the microdrop of the AgNPs solution was
55
56 applied on a suitable substrate and dried under the ambient conditions. Thus, the concentration of AgNPs in
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58 the microdrop is slowly increased. During the evaporation of water the fluctuations of the concentrations of
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small particles could be formed on the inhomogeneities of the substrate and such fluctuations led to the
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61 uneven coagulation of small nanoparticles and, respectively, to the formation of large (~10–40 nm) particles
62 9
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 of irregular shape. Based on the TEM-images the histograms of particles size distribution (PSD) were plotted
(Fig. 8, b, Figs. S2 – S5, see Supplemental Information). Presented PSD histograms are based on the analysis
1
2 of 3 to 6 TEM-images (120–150 particles) for each of the conditions of the experiment.
3
4 Table 2. Dependency of the size of obtained AgNPs on the reaction conditions
5
6 The fraction of particles
7 [Ag+]0104, mol/L C(RL), g/L d±d, nm
8
with a size within 1–4 nm, %
9 0.2 2.6±1.4 70
10
11 0.5 4 2.6±1.3 70
12
13 1 2.6±1.2 75
14
15 1 4.8±4.3 40
0.2
16 2 2.6±2.2 60
17
18
19 It was found (Fig. 8, b and Table 2) that the fractions of particles of 1–4 nm in size are more than 70 %
20 for the C(RL) = 4 g/L. At the same time, the distribution of large (above 10 nm) particles is nonmonotonic
21
22 and cannot be described even using of polymodal distribution functions. These facts indirectly confirm the
23
24 above mentioned assumption about the coagulation of particle during preparation of the sample for TEM
25 investigations, because, in the case of coagulation of AgNPs at the stage of their growth the distribution of
26
27 the obtained AgNPs should be more monotonous and PSD should be described by the Gaussian function.
28
29 As it can be seen from the presented in Table 2 data, an increase of the concentration of silver ions has
30 almost no effect on the size of the resulting AgNPs: the values of their average diameter (d) and
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32 polydispersity (d) are close. At the same time, with the decrease of the RL concentration to 2 g/L, an
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34 increase of d AgNPs is observed, and with a further decrease of the stabilizer concentration, together with
35
36 d, the value of the average particle diameter is also increased.
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38 3.3. Analysis of the kinetics of AgNPs formation in RL solutions.
39
40 Based on the obtained experimental data the following generalizations and assumptions which are
41 necessary for further analysis of the mechanism of formation of AgNPs in RL solutions can be done:
42
43 i) the kinetic curve of AgNPs formation is characterized by a pronounced induction period and is
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45 sigmoidal which indicates, that the reduction of Ag+ in RL solutions is autocatalytic process;
46 ii) the order of reaction with respect to RL is equal to 1; therefore, given that the concentration of RL
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48 in the solution in the all of cases is much higher than the concentration of silver ions, the “gross reaction” of
49
50 the formation of AgNPs can be represented as Ag+  Ag0;
51 iii) due to the high activation energy of the nucleation and the growth the diffusion nature of the
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53 process of nucleation of AgNPs is unlikely, i. e. the limiting stages of nucleation and growth are the chemical
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55 processes of reduction of silver ions in solution (nucleation) or on the surface of AgNPs (growth);
56 Therefore, the kinetic curves of AgNPs formation in rhamnolipid solutions were analyzed using the
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58 different Finke-Watzky schemes, namely (1.1) FW-2 (Watzky and Finke. 1997), (1.2) FW-3 (Widegren et
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 al. 2001), (1.3) FW-4 (Besson et al. 2005) and (1.4) FW-4a (Finney and Finke. 2008; Finney et al. 2012;
Özkar and Finke. 2016):
1
2 A k  B
1,obs

3 (1.1)
4 A  B 
kg
2B
5
6 A k  B
1, obs

7 A  B 
kg
2B (1.2)
8
9 B  B 
k3
C
10
11 A k  B
1, obs

12
A  B 
kg
2B
13 (1.3)
14 B  B 
k3
C
15
16 B  С 
k4
1,5
17
18 A k  B
1, obs

19 A  B 
kg
2B
20 (1.4)
21 B  B 
k3
C
22
23 A  С 
k4
1,5C
24
25
Here A is a precursor, B is a metal atom in the nanoparticle and C is a metal atom in the agglomerated
26 nanoparticle.
27
28 The systems of ordinary differential equations (ODE) for such schemes are written as follow:
29
dA
30   k1, obs A  k g AB
31 dt
(2.1)
32 dB
33  k1, obs A  k g AB
34 dt
35 dA
36  k1, obs A  k g AB
37 dt
38 dB
39  k1, obs A  k g AB  2k3 B 2 (2.2)
dt
40
dC
41  k3 B 2
42 dt
43
dA
44  k1, obs A  k g AB
45 dt
46 dB
47  k1, obs A  k g AB  2k3 B 2  k4 BC (2.3)
48 dt
49 dC
50  k3 B 2  0.5k4 BC
dt
51
52 dA
53  k1, obs A  k g AB  k4 AC
54
dt
dB
55  k1, obs A  k g AB  2k3 B 2 (2.4)
56 dt
57 dC
58  k3 B 2  0.5k4 AC
59 dt
60
61
62 11
63
64
65
 The FW-2 scheme is usually used to describe the kinetics of the metal nanoparticles formation in
solutions in the absence of the agglomeration of particles. At the same time, the schemes FW-3, FW-4 and
1
2 FW-4a take into account the processes of the nanoparticle aggregation (2.2), secondary agglomeration of
3
4 particles (2.3), as well as the autocatalytic growth of agglomerates (2.4). In Table 3 there are observed rate
5 constants of the corresponding reactions obtained by optimization of the experimental kinetic curves.
6
7
Table 3. Calculated values of the observable rate constants of the pseudo-elementary reactions of the AgNPs
8
9 formation in solutions of rhamnolipid
10
11 [Ag+]0, mol/L [RL], g/L Parameter FW-2 FW-3 FW-4 FW-4a
12
–1
13 k1,obs, min 0.0032 0.004 –– ––
14
15 kg, mol–1·L·min–1 1890 1800 –– ––
16
17
210–4 k3, mol–1·L·min–1 –– 190 –– ––
18 k4, mol–1·L·min–1 –– –– –– ––
19
20 RMS 4×10–6 4×10–6 –– ––
21
22 k1,obs, min–1 0.006 0.004 –– ––
23 kg, mol–1·L·min–1 1260 1260 –– ––
24
25 510–4 4 –1
k3, mol ·L·min –1
–– 200 –– ––
26
27 k4, mol–1·L·min–1 –– –– –– ––
28
29 RMS 6×10–6 6×10–6 –– ––
30 k1,obs, min–1 0.008 0.007 –– ––
31
32 kg, mol–1·L·min–1 1300 1350 –– ––
33
34 1010–4 k3, mol–1·L·min–1 –– 0.01 –– ––
35
k4, mol–1·L·min–1 –– –– –– ––
36
37 RMS 1×10–5 1×10–5 –– ––
38
–1
39 k1,obs, min 0.007 0.0016 0.0016 0.0014
40
41 kg, mol–1·L·min–1 830 1650 1680 1800
42 1 k3, mol–1·L·min–1 –– 960 980 2050
43
44 k4, mol–1·L·min–1 –– –– 0 540
45
46 RMS 8×10–6 4×10–6 4×10–6 3×10–6
47 210–4
k1,obs, min–1 0.004 0.0023 0.0023 0.0016
48
49 kg, mol–1·L·min–1 1300 1660 1650 1900
50
–1 –1
51 2 k3, mol ·L·min –– 440 430 1500
52
53 k4, mol–1·L·min–1 –– –– 0 1200
54 RMS 5×10–6 2.6×10–6 2.6×10–6 2×10–6
55
56
57 It was found that at a concentration of RL equal to 4 g/L the kinetic curves are satisfactorily described
58
59 by the scheme FW-2. As it can be seen from the presented in Table 3 data, calculated according to the
60
schemes FW-2 and FW-3 values of k1,obs and kg almost coincide in the range of initial concentrations of
61
62 12
63
64
65
 silver nitrate within 0.0002 – 0.001 mol/L; the values k3 are low compared to values kg and the values of the
root mean square deviation (RMS) for the studied cases don’t depend on the selected model. Thus, the
1
2 analysis of the kinetics of AgNPs formation at different initial concentrations of AgNO 3 confirms the above
3
4 mentioned assumption about the absence of agglomeration of the particles at the stage of their growth.
5 However, with the decrease of RL concentration the polydispersity of the obtained particles as well as
6
7 their average diameter increase (Table 2); i.e. the agglomeration processes in these systems are probable.
8
9 This assumption is in good agreement with the results of the calculations presented in Table 3. As we can
10 see, the values of RMS are reduced by half in the case of using of the FW-3 scheme for the optimization of
11
12 kinetic curves of AgNPs formation at different RL concentrations and the lowest values of RMS are
13
14 observed for the FW-4a scheme.
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31 Fig. 9. Calculated by the schemes FW-2 (dashed line) and FW-4a values of concentrations of A, B
32
33 and C. Points are the experimental data obtained at the different concentrations of RL.
34
35 As it can be seen from Fig. 9, the kinetic curves are well described using the scheme FW-4a. This
36
37 means, that the formation of AgNPs at low concentrations of rhamnolipid occurs through the stage of
38 formation of agglomerates of particles with their subsequent autocatalytic growth. It should be noted that the
39
40 values of the observed rate constants of nucleation, growth, aggregation and secondary autocatalytic growth
41
42 of AgNPs cannot be directly used to estimate the size and concentration of the obtained nanoparticles due to
43 the pseudo-homogeneous approximation used in the all FW models. However, it is necessary to pay
44
45 attention to the values of the rate constants of coagulation (k3) and the rate constants of secondary
46
47 autocatalytic growth (k4) of nanoparticles at RL concentrations of 1 and 2 g/L. The value of k3 for a low
48 concentration of RL is higher compared to kg and k4, which indicates the predominance of the coagulation
49
50 processes in the reaction mixture. However, increasing of the concentration of RL to 2 g/L leads to the sharp
51
52 increase of the value of k4 and decreasing of k3. Hence, in this case the processes of growth (both primary
53
and secondary) of particles are predominant. The above considerations are in good agreement with the
54
55 results of TEM investigations of the obtained AgNPs (Table 2). The decrease of the concentration of RL in
56
57 the reaction mixture to 2 g/L leads to the increase of the polydispersity of the particles caused by the
58
secondary autocatalytic growth of agglomerates (k4), but the average diameter of AgNPs does not change.
59
60 Further decrease of RL concentration up to 1 g/L leads to the fact that the concentration of surfactant in
61
62 13
63
64
65
 solution becomes insufficient for the effective stabilization of AgNPs and the agglomeration processes (k3)
predominate in the system. Respectively, simultaneously with increasing of polydispersity of AgNPs their
1
2 average diameter also increases.
3
4 3.4. Testing of the plug flow reactor for the synthesis of AgNPs
5
6 The scheme of the continuous tubular reactor used for the testing of possibility of production of the
7
“AgNPs-RL” solutions is presented in Fig. 1 (vide supra) and the main aim of this Section was to optimize
8
9 the rate of the reagent feed into the reactor to provide the plug flow regime and full conversion of precursor.
10
11 Taking into account the obtained results of kinetic investigations as well as TEM and XRD measurements
12
the optimal conditions for the synthesis of AgNPs may be considered as follow: t=70 0C, pH=10, C(RL)=4
13
14 g/L, [Ag+]0=110–3 mol/L. Such conditions provide the high rate of the reaction which is close to the rate of
15
16 some fast electrochemical processes of AgNPs formation, for example with the rate of microplasma
17
18 synthesis of AgNPs (Kuntyi et al. 2021).
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37 Fig. 10. UV-vis spectra of AgNPs solutions obtained in batch reactor (1) and in continuous tubular
38
39 reactor (2).
40
41 The completeness of Ag+ ions transformation was tested using UV-vis spectroscopy. It was found that
42
43 in our case the optimal rate of the reagent feed was ~1 mL/min of “Ag++RL” solution. Such rate provides the
44 complete conversion of Ag+ ions into the AgNPs. Аs we can see from Fig. 10 the UV-vis spectra of AgNPs
45
46 obtained at the final of the kinetic experiment and AgNPs obtained in tubular reactor are very close. Such
47
48 value of the reagent feed rate may be explained as follow. As it can be seen from Fig. 4(b), the reaction of
49 reducing of Ag+ ions by RL at above mentioned conditions have completed at ~10 min. Then, it is logical to
50
51 assume that the rate of the reagent feed is equal to the volume of reactor divided by the time of the reaction
52
53 currying out.
54
55
56
57
58
59
60
61
62 14
63
64
65
 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16 Fig. 11. A change of the color of reaction mixture during reaction in continuous tubular reactor.
17
18
19 Despite the relatively low rate of the flow of the reaction mixture, the plug flow regime in the reactor
20 was observed (Fig. 11). This fact may be caused by the relatively high value of the flow linear velocity in the
21
22 reactor (u=8 cm/min), which is determined by the ratio of the volumetric flow rate () and the tube
23
24 transverse cross sectional area (S):
25
u = /S (3)
26
27 Needless to say, that the constructed laboratory setup is only the prototype. However, we can assume
28
29 that the presented observation as well as the kinetic experiments may be considered as “the starting point”
30
31
for the development of the industrial reactors for the preparation of the solutions of AgNPs.
32
33 Conclusions:
34
35 The method of “green” synthesis of silver nanoparticles in rhamnolipid solutions is presented. It was
36
37 found that the average diameter of the obtained AgNPs is 2.6 nm and does not depend on the initial
38 concentration of Ag+ but is increased in twice with the decrease of the concentration of RL from 4 to 1 g/L.
39
40 The kinetics of the formation of AgNPs was studied in details. It was found that both of the rate of nucleation
41
42 and the rate of growth of AgNPs are extremely increased with the increase of pH and temperature. The
43 activation energies of the nucleation and of the growth processes are close and are equal to 135 and 140
44
45 kJ/mol respectively. The kinetic curves of AgNPs formation were fitted using different types of Finke-
46
47 Watzky schemes. It was found that at the concentration of RL equal to 1 g/L the mechanism of particles
48 formation occurs via the steps of agglomeration subsequent by the autocatalytic growth of formed
49
50 agglomerates. On the contrary, at the concentration of RL equal to 4 g/L the kinetic curves are well-fitted
51
52 using the FW-2 scheme. This fact confirms the absence of agglomeration of the particles on the stage of
53 AgNPs growth. The optimal conditions for the production of the solutions “AgNPs-RL” were established as
54
55 pH=10, temperature 70 C, C(RL)=4 g/L and initial concentration of Ag+ equal to 0.001 mol/L. A laboratory
56
57 model of a flow tubular reactor for the synthesis of “AgNPs-RL” colloidal solutions has been created and the
58
optimal flow rate of the reaction solution for providing of full transformation of precursors was established.
59
60
61
62 15
63
64
65
 Acknowledgments
This work was carried out with the partial financial support of the National Research Foundation of
1
2 Ukraine (“Design of polyfunctional nanostructured mono- and bimetals with electrocatalytic and
3
4 antimicrobial properties” Agreement 165/02.2020).
5 The authors are gratitude to Professor Olena Karpenko and her scientific group from the Department
6
7 of PhChFF InPOCC NAS of Ukraine for the provided samples of rhamnolipid.
8
9 The research was partially performed on the equipment of the Scientific Equipment Collective Use
10 Center: “Laboratory of Advanced Technologies, Creation and Physico-Chemical Analysis of a New Substances
11
12 and Functional Materials” (https://lpnu.ua/ckkno).
13
14 Some separated (the motivation of the investigations and short result discussion) results obtained by
15 the authors were partially presented as a poster on the International conference “Nanotechnology and
16
17 nanomaterials” (NANO-2021) (August 25-27, 2021, Lviv, Ukraine).
18
19 Conflicts of interest: On behalf of all authors, the corresponding author states that there is no conflict
20
21 of interest.
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Figure 1 Click here to access/download;Figure;Fig1.eps

water from the

circulation thermostat

working solution

RL+AgNO3
“AgNPs-RL” solution
solution

2 4
Figure 2 Click here to access/download;Figure;Fig2.EPS

2.0 445

440

435

, nm
1.5 430
Optical Density

max
425

420

415
1.0

410

0 50 100 150 200 250

t, min
0.5

0.0

300 400 500 600 700 800 900 1000 1100

, nm
Figure 3 Click here to access/download;Figure;Fig3.EPS

0.20
1.0 (a) 0.35
1/tind (b) 0.18
a 0.30 da/dt 0.16
0.8 0.14
0.25

-1
da/dt, min
0.12

-1
0.6 0.20

1/tind, min
0.10
0.15
0.08
0.4 pH=8
pH=9 0.10 0.06
0.2 pH=10 0.05 0.04
pH=11
0.02
0.00
0.0 0.00
0 50 100 150 200 250 7 8 9 10 11 12
t, min
pH
Figure 4 Click here to access/download;Figure;Fig4.EPS

1.0 (a) (b)

1.0
a a
0.8 0.8 1 - 0.2 mmol/L
3
2 2 - 0.5 mmol/L
3 - 1.0 mmol/L
0.6 0.6 1

0.4 C(RL) = 1 g/L

C(RL) = 2 g/L 0.4
C(RL) = 4 g/L
0.2
0.2

0.0
0.0
0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60
t, min t, min
Figure 5 Click here to access/download;Figure;Fig5.EPS

0.22 0.6 0.5

1/tind 0.14 1/tind
0.20 da/dt da/dt
0.5 0.4
0.12
0.18

-1

-1
-1

-1
da/dt, min

da/dt, min
1/tind, min

1/tind, min
0.16 0.10 0.4 0.3

0.14 0.08
0.3 0.2
0.12 0.06
0.2 0.1
0.10
0.04
-4 -4 -4 -4 -3
0 1 2 3 4 5 2.0x10 4.0x10 6.0x10 8.0x10 1.0x10
C(RL), g/L [AgNO3]0, mol/L
Figure 6 Click here to access/download;Figure;Fig6.EPS

0
80 C 0 0
1.0 70 C
0 (a) (b) 1/tind
a 0
60 C -1
da/dt
-1
0.8

ln(da/dt)
-2 -2

ln(1/tind)
0.6

0.4 -3 -3

0.2
0
-4 -4
50 C
0.0
-5 -5
0 50 100 150 200 250 300 0.00280 0.00285 0.00290 0.00295 0.00300 0.00305
-1
t, min 1/T, K
Figure 7 Click here to access/download;Figure;Fig7.EPS

Intensity, a.u.

AgNPs-RL

RL

30 35 40 45 50 55 60 65 70 75 80 85 90

2 , degree
Figure 8 Click here to access/download;Figure;Fig8.tif
Figure 9 Click here to access/download;Figure;Fig9.EPS

-4 -4
2.0x10 + C(RL)= 1 g/L 2.0x10 +
A (Ag ) A (Ag ) C(RL)= 2 g/L

Concentrations, mol/L
Concentrations, mol/L

-4 -4
1.5x10 1.5x10

-4
1.0x10
C -4 C
1.0x10

-5 B B
-5
5.0x10 5.0x10

0.0 0.0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60
t, min t, min
Figure 10 Click here to access/download;Figure;Fig10.EPS

12 1

10 2
Optical Density

0
300 400 500 600 700 800 900 1000

l, nm
Figure 11 Click here to access/download;Figure;Fig11.tif
Supplementary Material

Click here to access/download

Supplementary Material
Supplementary_Bazylyak_new.pdf
Author’s Response to Reviewers‘ Comments Click here to access/download;Author’s Response to
Reviewers‘ Comments;Cover_Lett_2_Editor.docx

Dear Editors,

Presented manuscript is submitted to the Special Issue of the journal

"Applied Nanoscience" devoted to the International conference
"Nanotechnology and nanomaterials" (NANO-2021). Due to the conference
was proceeded in combined mode (offline and online), our own poster was
placed on the web-site of the Conference. And it is logical that some
separate results (namely, the motivation of the investigations and short
result discussion) were presented on our own poster. Above mentioned
poster is not the publication, but only the partial presentation of our
obtained results in the work of the Conference. However, in accordance
with Editors recommendations, we have supplemented the
Acknowledgement as follow:

“Some separated (the motivation of the investigations and short result

discussion) results obtained by the authors were partially presented as a
poster on the International conference "Nanotechnology and
nanomaterials" (NANO-2021) (August 25-27, 2021, Lviv, Ukraine).”

The correction has been marked by yellow.

With kind regards,

Dr. Liliya Bazylyak

 Supplementary Information

SILVER NANOPARTICLES PRODUCED VIA A GREEN SYNTHESIS

USING THE RHAMNOLIPID AS A REDUCING AGENT AND STABILIZER

Bazylyak L. I.1, Kytsya A. R.1,3, Lyutyy P. Y.2,3, Pilyuk Ya. V.1,

Korets’ka N. I.1, Kuntyi O. I.2

1
Department of Physical Chemistry of Fossil Fuels of the Institute of Physical-
Organic Chemistry and Coal Chemistry named after L. M. Lytvynenko of the
National Academy of Sciences of Ukraine. Naukova Str. 3а, Lviv, 79060, Ukraine
2
Lviv Polytechnic National University, Bandery Str. 12, 79013, Lviv, Ukraine
3
Karpenko Physico-Mechanical Institute of the NAS of Ukraine. Naukova Str. 5,
Lviv, 79060, Ukraine

Corresponding author:
Liliya Bazylyak, 3a Naukova Str., Lviv, 79060, Ukraine,
e-mail: bazyljak.l.i@nas.gov.ua

1
 Fig. S-1. XRD–pattern of AgNPs stabilized by RL. Graphical representation of
the Rietveld refinement of the powder data (observed X–ray powder diffraction
pattern is represented by the dotted line, calculated and difference (bottom) by the
solid lines, vertical bars indicate the Bragg positions).

2
 (a) (b)
Fig. S-2. TEM-image (a) and histogram of particle size distribution (b) of
AgNPs obtained at 70 C and initial concentrations of RL and AgNO3 equal to 4 g/L
and 210–4 mol/L respectively.

(a) (b)
Fig. S-3. TEM-image (a) and histogram of particle size distribution (b) of
AgNPs obtained at 70 C and initial concentrations of RL and AgNO3 equal to 4 g/L
and 510–4 mol/L respectively.

3
 (a) (b)
Fig. S-4. TEM-image (a) and histogram of particle size distribution (b) of
AgNPs obtained at 70 C and initial concentrations of RL and AgNO3 equal to 1 g/L
and 210–4 mol/L respectively.

(a) (b)
Fig. S-5. TEM-image (a) and histogram of particle size distribution (b) of
AgNPs obtained at 70 C and initial concentrations of RL and AgNO3 equal to 1 g/L
and 210–4 mol/L respectively.