Simultaneous Biosynthesis of Silver Nanoparticles with Spirulina sp. LEB 18 Cultivation

Abstract

Effluents from the textile, photographic, and other industries release considerable amounts of toxic metal ions, such as silver. Conventional physico-chemical treatments do not completely remove heavy metals from effluents. New technologies are being studied to remediate industrial effluents from the environment, such as the use of microalgae. Studies of the microalgae biosynthesis of silver nanoparticles have been performed using biomass or culture medium without cells or specific bioproducts synthesized by the microalgae. In this work, the simultaneous biosynthesis of silver nanoparticles and microalgae growth is presented for the first time. Nanoparticles 20 nm in size were biosynthesized between the 5th and 11th day of culture. Thus, an industrial effluent harmful to the environment containing elemental silver can be bioremediated by using an innovative, efficient, and ecologically safe method, with the simultaneous production of microalgae biomass and silver nanoparticles with potential applications in several areas.

Introduction

Population growth, urbanization, and industrialization are causing global concern due to the excessive discharge of wastewater into the environment.¹ The accumulation of heavy metal ions in food chains can cause severe damage to human health through the consumption of contaminated water or food plants grown in soil contaminated with metals.² Silver in its ionic form (Ag⁺) poses a great toxicological threat to aquatic ecosystems.³

Free silver may be present in the environment, or silver may interact with other organic and inorganic binding agents. Its speciation is influenced by the physical and chemical properties of the environment, which will determine its potential toxicity.⁴ Silver ions are persistent in the environment and can be concentrated within marine and terrestrial organisms and microorganisms, as they can be transported through the cell membrane.⁵

Innovative processes involving metal industrial waste should be directed not only toward the removal of pollutants from the environment but also toward the transformation of this effluent into a new product of commercial interest. In this context, microalgae biotechnology provides processes that, in addition to the potential bioremediation of Ag-containing residues, can biosynthesize silver nanoparticles (AgNPs) depending on the species of algae used.^6,7

AgNPs biosynthesis by microalgae promotes the reduction of metal using low- or zero-toxicity reducing agents such as microbial enzymes or biochemicals with reductive or antioxidant properties present in photosynthetic organisms.⁸ The advantages of the production of AgNPs by microalgae over conventional processes are the efficiency of Ag bonding due to the high surface area of contact between the microorganism and the metal, the lack of secondary residues generated, the use of chemical reagents only as a source of nutrients from the culture medium, the rapid growth of algae, the possibility of scaling up the process and the reuse of biomass.^1,9

Some microalgae and cyanobacteria have been studied previously, and their capacity to biosynthesize AgNPs has been confirmed, including Anabaena sp., Cylindrospermopsis sp., Lyngbya sp., Limnothrix sp., Synechocystis sp., Synechococcus sp., Botryococcus sp., Chlamydomonas sp., Chlorella sp., Coelastrum sp. and Scenedesmus sp. In previous studies, centrifuged biomass (intracellular synthesis) or supernatant (extracellular synthesis) was used.¹⁰ AgNPs synthesis by phycocyanin extracted from Spirulina platensis, polysaccharides extracted from Scenedesmus sp. and S. platensis biomass proteins have been studied previously.^10,11

In our work, we investigated the simultaneous biosynthesis of AgNPs and microalgae growth. This eliminates previous steps in the biosynthetic process such as the separation of biomass and culture medium or the extraction of specific biocomposites. Simultaneous biosynthesis and culture also allows continuous biosynthesis, as the microalgae release more reducing components into the medium as they grow, allowing more elemental silver to be added and reduced.^12,13 According to the desired application, biosynthesized AgNPs can be used together with the microalgae biomass, making use of the properties of the nanoparticles and of the active agents in the algal cells. Thus, microalgae biotechnology is a promising alternative for Ag bioremediation with the simultaneous production of biomass and AgNPs, presenting the advantages of process sustainability and profitability on an industrial scale.^14,15

Materials and Methods

Microorganisms and Culture Medium

The microorganism used in this study was Spirulina sp. LEB 18, previously isolated from Mangueira Lagoon located in Santa Vitória do Palmar, Rio Grande do Sul, Brazil.¹⁶ Zarrouk medium was used for culture and maintenance of the inoculum and contained (in g/L): NaHCO₃, 16.8; NaNO₃, 2.5; K₂HPO₄, 0.5; K₂SO₄, 1.0; NaCl, 1.0; Mg.SO₄·7H₂O, 0.2; CaCl₂, 0.04; FeSO₄·7H₂O, 0.01; and EDTA, 0.08, plus 1.0 mL/L of solutions A₅ and B₆. Solution A₅ (in g/L) contained: H₃BO₃, 2.86; MnCl₂·4H₂O, 1.81; ZnSO₄·7H₂O, 0.222; CuCO₄·5H₂O, 0.079; and MnO₃, 0.015. Solution B₆ (in g/L) contained: NH₄VO₃, 22.86; KCr(SO₄)₂·12H₂O, 192; NiSO₄·6H₂O, 44.8; Na₂WO₄·2H₂O, 17.94; TiSO₄·H₂SO₄·8H₂O, 61.1; and Co(NO₃)₂·6H₂O, 43.98.¹⁷ The inoculum of Spirulina sp. LEB 18 was maintained in 1-L Erlenmeyer-type photobioreactors in a thermocontrolled chamber at 30°C, with illumination provided by daylight fluorescent lamps of 41.6 μmol photons m⁻²·s⁻¹ and a photoperiod of 12 h light/dark.

Microalgae Culture Conditions for AgNPs Biosynthesis

The inoculum of Spirulina sp. LEB 18 was centrifuged (Refrigerated Centrifuge CR22GIII, Japan) at 10,000 rpm for 20 min, and the cells were washed with milli-Q water to remove salts. Culturing was performed in 1-L, Erlenmeyer-type closed photobioreactors, maintained on an orbital shaker with incubation at 110 rpm and 30°C, with a 24-h light photoperiod. The initial biomass concentration of the cultures was 0.50 g/L, and experiments were performed in duplicate for 20 days and with 1 mM of silver sulfate solution (Ag₂SO₄). The original carbon source of the Zarrouk medium (NaHCO₃) reacted with Ag₂SO₄ and was replaced with 0.4 g/L of glucose. Three experiments were performed with different culture conditions: (1) Spirulina sp. LEB 18 with modified Zarrouk medium; (2) modified Zarrouk medium with Ag₂SO₄ solution; and (3) Spirulina sp. LEB 18 in modified Zarrouk medium with Ag₂SO₄ solution.

Analytical Determinations

Biomass concentrations were monitored daily and determined by optical density measurements of the cultures using a spectrophotometer at 670 nm (Spectrophotometer UVmini-1240, Shimadzu) and a standard curve that correlates optical density with biomass dry weight. The pH was monitored every 24 h (Universal Inducer pH 0–14, Merck, Germany).

Preparation of Samples for the Analysis of AgNPs

To assess the hydrodynamic diameter and zeta potential and perform transmission electron microscopy, culture samples were placed in an ultrasonic bath for 10 min, centrifuged at 6,000 rpm for 10 min (Refrigerated Centrifuge CR22GIII, Japan) and subsequently filtered through 0.22 μm membranes (Millipore, Bedford, MA) to separate the biomass from the culture medium. The resulting supernatant from this step contains intracellular and extracellular AgNPs.

Hydrodynamic Diameter and Zeta Potential of AgNPs

After the first stage of preparation, samples were diluted 1:20 in milli-Q water; subsequently, the hydrodynamic diameter (HD) and zeta potential (ZP) (Zetasizer Nano Malvern ZS90, Japan) were monitored every 24 h.

Morphology and Diameter of AgNPs

To determine the morphology and size of the AgNPs produced, analysis was conducted using a Transmission Electron Microscope (TEM) (Jem-1400, Japan) on the 5th and 11th days of culture. Samples free of microalgae cells, prepared according to above, were deposited on amorphous carbon film supported on a 3.0-mm diameter copper grid and then placed in a desiccator for 24 h to allow complete evaporation of the liquid.

Results and Discussion

The addition of Ag₂SO₄ to Spirulina sp. LEB 18 did not inhibit the growth of the microalgae. In fact, higher growth (2.68 g/L) was observed from the 5th to the 15th day of culture compared to tests performed without the added metal and sulfate (1.46 g/L) ( Fig. 1 ).

Fig. 1.

Growth curve of Spirulina sp. LEB 18 (control) (○); and Spirulina sp. LEB 18 with the addition of Ag₂SO₄ (■).

The presence of metal ions per se is thought to have nominal impact on microalgae growth because biosorption comprises the binding of metals to biomass, enzymes, or their bioproducts via a process that does not involve metabolic energy.¹⁸

The culture of Spirulina sp. LEB 18 with Ag₂SO₄ exhibited blue-green coloration since the beginning of cultivation ( Fig. 2a ) until day 4 ( Fig. 2b ), characteristic of microalgae, but changed to yellow on day 5 ( Fig. 2c ) and orange on day 11 ( Fig. 2d ). The yellow and orange colors obtained in this study are presumed to be due to surface plasmon resonance. This phenomenon occurs at the boundary of a metal on the nanoscale that is excited by an external electromagnetic field. The free electrons on the surface of the metal are induced by the electromagnetic field to generate a maximum oscillation at a certain frequency, resonating with the frequency of oscillation of incident light.¹⁹ Since localized surface plasmon resonance is sensitive to changes in the local dielectric environment, biomolecules chemically bonded at the interface of the silver particles result in the near-surface increase of the refractive index. Changes in refractive index lead to changes in surface plasma waves (surface plasma polarity), the original coloration of the solution changing to yellow. These changes are measured in real time resulting in a change in ʎ_max. This property can be employed to detect changes in the size of the nanoparticles by monitoring the wavelength change measurement.

Fig. 2.

Change in cultivation coloration of Spirulina sp. LEB 18 with solution of Ag on (a) day 1; (b) day 4; (c) day 5; and (d) day 11. Color images are available online.

Thus, colloidal solutions of AgNPs are yellowish in color. This resonance frequency is directly linked to the shape, size, organization of the nanoparticles, and the refractive index of the medium.²⁰ The change in color of the cultures on day 5 to yellow and then to orange indicated the early formation of AgNPs ( Fig. 2d ).

UV-Vis spectrum readings showed absorbance peaks around 450 nm ( Fig. 3b,c ). According to Solomon et al., AgNPs of 5–10, 10–14, 35–50, and 60–80 nm in diameter exhibit peaks at wavelengths of 380–390, 395–405, 420–435, and 438–450 nm, respectively.²¹

Fig. 3.

UV-vis spectra of AgNPs biosynthesized by Spirulina sp. LEB18 on (a) day 11; (b) day 5; and (c) day 0 of culture.

Thus, in TEM analysis we confirmed this affirmation when we found particles with 80 nm obtained from the extracellular and intracellular medium. Moreover, TEM analyses performed at the 5^th and 11th days of culture confirmed the formation of Ag nanoparticles ( Fig. 4 ).

Fig. 4.

Transmission Electron Micrographs showing the presence of biosynthesized AgNPs on (a) day 5 and (b) day 11 of culture of Spirulina sp. LEB 18 with added Ag₂SO₄.

The polydispersity index, which provides information on the homogeneity of the size distribution of particles, was 1.0 for the culture of Spirulina sp. LEB 18 with Ag₂SO₄, indicating the formation of a broadly polydisperse system.^22,23

On day 5, when the cultures turned yellow, the predominant size of AgNPs, obtained intra- and extracellularly, was approximately 70 nm, and at that time, the nanoparticles present ranged in size from 40 to 160 nm ( Fig. 5a ). On day 11 of culture, both the size range and diameter of the nanoparticles decreased, and we observed AgNPs between 10 and 80 nm, with a predominance of nanoparticles approximately 20 nm in diameter ( Fig. 5b ). It can be explained because in the exponential phase (from day 4 to day 8 of cultivation), the reduction may have been due to metabolites excreted in the extracellular medium. The size reduction continued in the cell death phase (up to day 11 of culture), where it is believed that the intracellular metabolite has been released into the extracellular medium through the disruption of cells, which may have occurred due to exhaustion of nutrients from the culture medium and thus loss of biochemical viability of the cell. After day 11, the culture entered the death phase and did not have viable cells for the production of reducing metabolites. For a possible identification of the functional groups that contributed to the formation of AgNPs, it would be necessary to perform Fourier Transform Infrared Spectroscopy in future studies.

Fig. 5.

AgNPs size distribution on (a) day 5 and (b) day 11 of culture of Spirulina sp. LEB 18 grown with Ag₂SO₄.

According to research by Patel et al., the reduction of silver particles to AgNPs can be performed in cell-free liquid culture or in the presence of microalgae biomass, and AgNO₃ was shown to be toxic to microalgae when cultured together.¹⁰ However, in our study, Spirulina sp. LEB 18 was grown in the presence of Ag₂SO₄, during which time the reduction of silver particles to AgNPs occurred. This may have been possible due to the neutralization of the charges present on the Ag salts by extracellular compounds produced by the microalgae.^10,24

The mechanism of AgNPs reduction biosynthesis consists of the capture of Ag⁺ particles by electrostatic interactions, through negatively charged biomolecules, produced intracellularly by microalgal cells, that can be found inside the cell or excreted into the extracellular medium.²⁵ Neutralization occurs by transferring an electron from the oxidizing agent to the reducing agent (terpenoids, carbonyl groups, phenolics, flavonones, amines, amides, proteins, pigments, alkaloids, nicotinamide adenine dinucleotide phosphate), forming the nanoparticles in the aqueous phase or at the interface of the reducing molecules.^10,26 The reduced atoms form stable nuclei. However, in some cases, the aggregation process may occur when the colloid is not stable enough. Thus, the nanoparticles approach in order to agglomerate.²⁷

However, in this study, as biosynthesis is simultaneous to microalgal growth, steric stabilization is thought to occur, where adsorption of polymeric additives occurs around the nanoparticles, so that the polymer chains prevent the nanoparticles from approaching. Therefore, the cultivation of microalgae simultaneously to the biosynthesis of nanoparticles allows the process to occur continuously. As the microorganism grows, more active compounds are released in the medium. Consequently, more elemental silver is reduced to nanoparticles.

The zeta potential values for the experiments performed without the addition of microalgae were lower than 30 mV ( Fig. 6 ), and in the experiments containing Spirulina sp. LEB 18, values greater than 30 mV were obtained within a few days. According to Belin et al., colloidal suspensions are considered stable based on the zeta potential measurements of the particles being above +30 mV or below −30 mV.¹⁸ This finding indicates that the colloidal suspension of AgNPs in microalgae culture was stable on days 12, 13, and 14 and that the charge distribution on the surface of the particles was negative.

Fig. 6.

Zeta potential (mV) of the particles in culture with Ag₂SO₄ without addition of microalgae (□) and with Spirulina sp. LEB 18 (■).

According to Prabhu and Poulose, the factors responsible for the synthesis of AgNPs are the microbial enzymes or phytochemical compounds with reductive or antioxidant properties present in photosynthetic organisms, such as Spirulina, which act on precursors.⁸ According to Govindaraju et al., the intracellular biosynthesis of AgNPs occurs through the presence of S. platensis dry biomass proteins, which are responsible for bioreduction.¹¹ Proteins are capable of binding nanoparticles through amino acid residues such as cysteine or through free amine groups, thereby promoting their stabilization.²⁸

In this study, the reduction of silver may have involved more than one microalgae bioproduct, as there was no separation of culture liquid and microalgae biomass. Thus, biosynthesis simultaneous to microalgae culture can increase the concentration of AgNPs produced in addition to reducing process costs because there is no need for biomass-medium separation processes or the extraction of specific biocomposites.

Thus, this study shows a promising alternative for silver bioremediation. The microalgal cells were able to absorb the metal and biosynthesize AgNPs through produced biomolecules, demonstrating the possible efficiency to remove significant concentrations of this metal.

Conclusion

In this study, the biosynthesis of AgNPs was performed simultaneously with microalgae growth and was shown not to require the separation of the biomass and culture medium or the extraction of biomass bioproducts. AgNPs with 20 nm of diameter were biosynthesized by the microalgae Spirulina sp. LEB 18 between the 5th and the 11th days of culture, with maximum algal cell concentrations reaching 2.68 g/L. The biosynthesis of AgNPs simultaneously with microalgae cultivation is an innovative method for the bioremediation of heavy metals with the production of microalgae biomass and the biosynthesis of nanobiomaterials that can be applied in several areas.

Footnotes

Acknowledgments

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. The authors also acknowledge CNPq (National Council of Technological and Scientific Development) and MCTIC (Ministry of Science Technology, Innovation and Communications).

Author Disclosure Statement

No competing financial interests exist.

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