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PII: S0141-8130(17)32898-2
DOI: https://doi.org/10.1016/j.ijbiomac.2017.12.007
Reference: BIOMAC 8668
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BIOSYNTHESIS OF SILVER NANOPARTICLES AND
IN ANTIMICROBIAL APPLICATIONS
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a
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Novel Materials and Nanotechnology Group, Institute of Agrochemistry and Food
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b
UCIBIO-REQUIMTE, Chemistry Department, Faculty of Sciences and Technology,
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Universidade NOVA de Lisboa, Campus da Caparica, Caparica, Portugal
c
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Polymer Biotechnology Group, Biological Research Center (CIB-CSIC), CSIC, 280140,
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Madrid, Spain
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*
Corresponding author. IATA-CSIC, Avda. Agustín Escardino, 7, 46980, Paterna,
ABSTRACT
This study deals with the optimization and scaling up of the production of poly(3-
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of the chemical and biological synthesis of AgNPs during the fermentation process of
demonstrated the inherent capacity of C. necator to reduce the silver salt and produce
AgNPs without the need for adding a reducing agent and, that the method of synthesis
(with or without reducing agent) affects the dispersion of the AgNPs and their
and the relevant physical properties of the PHB-AgNPs nanocomposites pressed into
films were determined. From the characterization work, the AgNPs were found to be well
dispersed and distributed into the polymer matrix, having a maximum frequency of
particles with average diameter of 76-95 nm. Moreover, the presence of AgNPs did not
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cause any effect on the thermal properties of the biopolymer, although a slight reduction
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in crystallinity was seen. The developed materials presented a strong antimicrobial
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activity against the food-borne pathogens Salmonella enterica and Listeria
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monocytogenes, which makes them potentially suitable for active coatings and packaging
INTRODUCTION
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Given the environmental concerns related to the massive use of petroleum-based plastics
and their high demand in the packaging market, which was around 40% of the global
obtained from renewable resources has emerged in the last decades. Among the most
[2].
PHAs are synthesized by many bacteria as intracellular carbon and energy storage
granules. The Gram negative, facultative bacterium Cupriavidus necator (before called
Ralstonia eutropha and Wautersia eutropha) has been intensively studied for almost 50
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years [3]. C. necator was described by [4] as a non non-obligate bacterial predator of
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various soil bacteria and fungi ([5-7] and it is currently an interesting candidate for the
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necessary characteristics, which are required for a biotechnological production strain.
Due to its metabolic versatility, it can convert a broad range of renewable carbon
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resources into diverse valuable compounds [8]. It uses fragmented organic wastes
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(conversion of organic wastes to short chain volatile fatty acids through hydrolysis and
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acidogenesis) from the food and beverage industries for the PHAs production [9].
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The PHB produced by C. necator can be used for applications ranging from replacement
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packaging field, PHB has been used to make various biodegradable products, including
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films, coated paper and boards, compost bags, dispensable food service-ware, and molded
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Particularly, packaged foods with enhanced quality and safety are demanded by
high concentrations of antimicrobials in the food matrix to extend foods shelf-life [12].
Inorganic compounds in the nanosize range, i.e. below 100 nm, such as silver
nanoparticles (AgNPs) have emerged as efficient tools to prevent microbial spoilage and
microbial outbreaks in food contact plastics and surfaces. Traditionally, AgNPs are
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synthesized by chemical methods using different organic and inorganic reducing agents,
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such as sodium borohydride (NaBH4) [13, 14], sodium citrate [15], ascorbate [16],
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elemental hydrogen, Tollen’s reagent, N,N-dimethyl formamide (DMF) and
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poly(ethylene glycol) block copolymers [17]. However, most chemicals used in this kind
of synthesis are toxic and lead to non-eco-friendly by-products. To solve these problems,
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biological synthesis of nanomaterials using living organisms such as bacteria [18], fungi
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[19] or plant extracts [20] are currently being developed.
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So far, there is only a study reported in the literature about the effects of AgNPs on C.
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necator growth dynamic, which indicates growth stimulation of this bacteria at certain
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AgNPs concentrations (40µg/mL of AgNPs with primary particle size distribution D90 <
nanoparticles at different growth stages [21]. However, because it has been known and
accepted that the silver susceptibility depends on the medium culture composition [22],
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produced. On the other hand, in a recent publication, the synthesis of AgNPs was carried
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out by using the cell-free supernatant of Alkaliphilus oremlandii, strain ohILA, culture.
In that case, the synthesized AgNPs were subsequently stabilized by the extracted and
of a simple bioprocess for the simultaneous biosynthesis of PHB and AgNPs with
synthesis of AgNPs during the fermentation process of C. necator at shake flask was
carried out and then optimized and scale-up to fully-automated 10 liters bioreactor.
Moreover, the physical properties and the antimicrobial performance of the developed
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nanocomposites, was determined.
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2.0 MATERIALS AND METHODS
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2.1 Materials
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Spain), chloroform stabilized with ethanol (Technical grade, Panreac, Spain), methanol
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(For analysis, ACS, ISO, Panreac, Spain), hiperpuric nitric acid (69%, Panreac, Spain),
sulphuric acid in methanol solution (Sigma–Aldrich, HPLC grade) and benzoic acid in
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chloroform (1 g/L) (Sigma–Aldrich, HPLC grade) without further purification were used
Cupriavidus necator strain H16 (DSM 428, Wilde 1962) was obtained from the German
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Collection of Microorganisms and Cell Cultures (DSMZ) and used in this study. The
culture, cryopreserved in 20% (v/v) glycerol at -80 °C, was reactivated in solid Luria
cultivation medium consisted of: MgSO4.7H2O, 0.39 mg/L; K2SO4, 0.45 mg/L;
FeSO4.7H2O, 15 mg/L; NH4Cl, 0.01% (w/v); H3PO4, 1 M (12 mL/L); trace elements, 24
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patent pending methodology (Spanish Patent P201630829). First, the process was
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analyzed at shake flask scale and then optimized and scaled-up to 10 L capacity fully-
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automated laboratory-scale bioreactor.
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2.3.1 Synthesis at shake flask scale
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Batch cultivation experiments of C. necator in shake flask were performed in order to
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investigate the AgNPs synthesis from silver nitrate. The inoculum for shake flasks
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medium and incubation in an orbital shaker at 30° C and 200 rpm for 24 h. The cells were
then harvested and resuspended in the culture medium described above (see 2.2 section)
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to reach an optical density of 0.3 at a wavelength of 600 nm. The cultivation was done in
2 L Erlenmeyer flask with an initial working of 800 mL, which were incubated at 30° C
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and 200 rpm for 24 h. Subsequently, the cultureswere treated with 267 mL of silver nitrate
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solutions at 1 mM or 3.5 mM. The silver nitrate treatment was applied at the beginning
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and after 24 h of the cultivation and carried out both in presence and in absence of a
reducing agent. For the experiments in the presence of the reducing agent, 8 mL of the
concentration and, after 20 min, the desirable silver nitrate solution were added dropwise
During the addition of silver nitrate and sodium borohydride and for further 16 h, the
Sartorius, Germany), with an initial working volume of 7 L. The inoculum was 10% (v/v)
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of the initial reactor working volume. It was prepared by inoculating a C. necator colony
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into LB medium and incubation in an orbital shaker, at 30° C and 200 rpm, for 24 hours.
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The culture thus obtained was then transferred into culture medium described above (see
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2.2 section), further incubated for 48 h at the same agitation and temperature and used for
the bioreactor experiments. The bioreactor was operated with controlled temperature (30
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°C) and pH (7.0). The pH was controlled by the automated addition of HCl (1 M) and
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NH4OH (25% v/v, in the first 20 hours) which served also as nitrogen source, or NaOH
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(2 M, after 20 hours) to impose nitrogen-limitation conditions. The dissolved oxygen
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(DO) concentration was controlled at 30% by automatically adjusting the stirring rate
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(between 300 and 2000 rpm). The experiments comprised an an initial batch phase of 20
L/h were fed to stimulate the polymer accumulation. At the end of the cultivation runs, a
sample of 10 mL was withdrawn from the bioreactor of broth was centrifuged at 4000
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rpm for 15 min and the pellet washed with deionized water and lyophilized for the
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gravimetric the cell dry weight (CDW) quantification. In the bioreactor experiments the
silver nitrate treatment was applied following the optimal parameters obtained in the
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was carried out by using organic solvents. To this end, the culture broth was centrifuged
for 15 min at 4000rpm, the supernatant discarded and the biomass transferred to 300 mL
closed bottle with stirred with a magnetic bead. Subsequently, 300 mL of distilled water
was added to the polymer rich solution. The mixture was separated in volumes of 50 mL,
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stirred in vortex for 1 min and centrifuged for 2 min at 2000 rpm to separate the organic
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phase. The resulting organic fraction was removed using glass Pasteur pipettes, and the
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polymer was then precipitated in ice-cold methanol (10-fold excess) with vigorous
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stirring. Finally, the precipitated polymer was separated by filtration and dried overnight
in a vacuum oven at 60 °C. The PHB content in the biomass (%, w/w) and the overall
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volumetric productivity (rp, g/ L day) was were calculated using the equations 1 and 2,
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respectively following equation (Equation 1):
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∆𝑃
𝑟p = (2).
∆𝑡
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where P (g /L) is the PHB produced during cultivation time t (day). The purity of the
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[24, 25]. Briefly, 3-5 mg of the extracted polymer were hydrolyzed with 20% (v/v)
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sulfuric acid in methanol solution (1 mL) and benzoic acid (internal standard) in
chloroform (1 mL), at 100 °C for 3.5 h. After the digestion, the methylated monomers
were extracted and injected into a gas chromatograph (Varian CP-3800) coupled with a
1 mL/min. The oven temperature program was as follows: 40 °C; 20 °C/min, until 100
°C; 3 °C/min, until 175 °C and, finally, 20 °C/min, until 220 °C. The injector and detector
temperature were set at 280 °C and 250 °C, respectively. Pure copolymer solution (0.25-
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2.5 Transmission electronic microscopy (TEM) of bacteria
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The size and morphology of AgNPs obtained from the shake flask experiments were
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characterized using a transmission electron microscope (JEOL l010, Hitachi) equipped
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kV. The samples for TEM analysis were prepared by fixing the cells with 2.5%
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glutaraldehyde and 2% osmium tetraoxide for 2 h at room temperature. The pellets were
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then washed three times with phosphate buffer saline (PBS) and dehydrated in ethanol
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series (30%, 70%, 90% and 100%). The specimens were further embedded in LR-White
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resin and cut with an ultramicrotome (Leica UC6). Ultrathin sections were then positively
stained with uranylacetate and lead citrate and then placed onto carbon-coated copper
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The polymer produced in the lab-scale reactor experiments was compression molded into
films of 80 µm of thickness using a hot-plate hydraulic press (Carver 4122, USA) at 180
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°C and 1.8 MPa for 5 min and used for further characterization. Pure and silver containing
films were prepared for comparative purposes. The morphology and size distribution of
AgNPs into the film were analyzed through observation of ultramicrotomed cross
sections by TEM using the same microscope that was used above (JEOL l010, Hitachi).