Green Synthesis of Gold and Silver Nanoparticles Using Plant Extracts, Fungal Filtrates, and Bacterial Cultures

Summary

This protocol describes reproducible green synthesis methods for fabricating gold and silver nanoparticles using plant extracts, fungal filtrates, and bacterial cultures, followed by nanoparticle confirmation through ultraviolet–visible spectroscopy and transmission electron microscopy.

Abstract

Green synthesis of metal nanoparticles (NPs) using biological systems provides a sustainable alternative to conventional chemical fabrication methods. This protocol presents reproducible experimental and analytical procedures for the synthesis of gold (Au) and silver (Ag) NPs using bacterial cultures, fungal filtrates, and plant leaf extracts as reducing and stabilizing agents. AgNPs were synthesized using extracts from Psidium guayaquilensis, Acanthophora spicifera, and Earliella sp., exhibiting characteristic ultraviolet–visible (UV–Vis) absorption bands between 405 and 425 nm that confirmed NP formation. A design-of-experiments approach was implemented to evaluate the influence of environmental factors, including oxygen conditions, pH, Au concentration, cell concentration, electron donor type, and temperature, on the synthesis of AuNPs by Shewanella oneidensis and Cupriavidus metallidurans. Under ideal conditions (0.2 mM Au and pH 5), S. oneidensis produced predominantly spherical AuNPs with an average size of 43.6 ± 11.0 nm and a characteristic absorption peak at 520 nm. NP formation and morphology were confirmed using UV–Vis spectroscopy and transmission electron microscopy. This workflow provides a reproducible platform for biogenic NP synthesis and supports applications in biosensing, bioremediation, and antimicrobial technologies.

Introduction

Metallic nanoparticles (NPs) possess unique physicochemical properties associated with their high surface area, including distinctive electronic, magnetic, and catalytic behaviors, as well as high reactivity. These characteristics have promoted their application across multiple fields, including medicine for disease diagnosis and drug delivery^1,2,3,4,5,6, cosmetics for sunscreen formulations⁷, electronics for transistors and solar cells⁸, environmental engineering for water treatment^2,9,10, and agriculture for fertilizers and pesticides^11,12. The continuous expansion of NP applications across disciplines is reflected in the increasing number of related publications reported in recent years^13,14.

Synthetic methods for metallic NPs enable precise control over important characteristics such as particle size, morphology, and stability, making the synthesis process a critical determinant of NP performance. Conventional chemical synthesis methods have been extensively optimized; however, they frequently involve toxic reagents, high energy consumption, and elevated production costs^14,15. These limitations have motivated the development of greener and more sustainable alternatives. Green synthesis approaches employ biological systems such as plant extracts ^16,17, microorganisms including fungi and bacteria¹⁸, and algae¹⁹ as natural reducing and stabilizing agents. During these processes, metal salts are reduced to zero-valent forms, generating NPs through environmentally friendly and cost-effective pathways. In addition, biologically synthesized NPs are often biocompatible, facilitating their use in medical, agricultural, and environmental applications^8,14.

The selection of an appropriate biological route for NP synthesis depends on the intended application, as well as the available infrastructure and operational requirements. Plant-based synthesis is generally preferred for rapid and low-cost NP production because it requires minimal sterile handling; however, variability in extract composition may affect reproducibility^20,21. Fungal-mediated synthesis can provide high NP yields and extracellular enzymes that improve NP stabilization and downstream processing, although this approach requires longer cultivation periods and controlled growth conditions²². In contrast, bacterial systems enable tunable synthesis under defined metabolic conditions and rapid biomass production, making them suitable for mechanistic studies and NP-associated applications²². However, bacterial synthesis often requires strict aseptic handling, controlled bioreactor conditions, and additional downstream processing when intracellular NPs are recovered^21,22.

Despite the advantages mentioned, green synthesis methods continue to face challenges associated with reproducibility and standardization. Furthermore, detailed visual step-by-step protocols for the green synthesis of metallic NPs remain limited²³, restricting the ability of researchers to reproduce and implement existing methodologies successfully. The goal of this work is to present reproducible green synthesis protocols for gold (Au) and silver (Ag) NPs using plant extracts, fungal filtrates, and bacterial cultures. Specifically, aqueous extracts from Psidium guayaquilensis and Acanthophora spicifera, extracellular fungal filtrates from Earliella sp., and bacterial systems involving Shewanella oneidensis and Cupriavidus metallidurans were employed for NP synthesis. The presented protocols emphasize critical steps in biological extract preparation, NP synthesis, and preliminary NP characterization. In addition, the workflow integrates design of experiments, standardized preparation procedures, and in-line characterization to support reproducible and optimizable biogenic NP synthesis.

Protocol

The research was performed in accordance with the institutional guidelines of the Escuela Superior Politécnica del Litoral (ESPOL, Ecuador). The plant, algal, and fungal biomass used in this study were collected from accessible environments and did not involve protected species or restricted areas. According to institutional and national regulations, no specific permits were required for the collection of freely available biomass under these conditions. Refer to Figure 1 for the integrated experimental workflow used for green synthesis of AgNPs and AuNPs using plant, algal, fungal, and bacterial biological systems.

Figure 1: Integrated Workflow for Green Synthesis of Silver (Ag) and Gold (Au) Nanoparticles (NPs) Using Biological Systems. Schematic of the experimental workflow for the green synthesis of AgNPs and AuNPs using plant, algal, fungal, and bacterial systems. The workflow illustrates biological source selection, preparation of extracts or biomass, reaction with metal precursors (AgNO₃ or HAuCl₄), reaction conditions, and NP formation. The biological systems include Psidium guayaquilensis leaf extract, Acanthophora spicifera algal extract, Earliella sp. extracellular filtrate, and Shewanella oneidensis MR-1 bacterial cultures. Please click here to view a larger version of this figure.

NOTE: Follow all institutional laboratory safety guidelines when handling chemicals and biological materials. Consult safety data sheets for all reagents before use.

1. Synthesis of AgNPs Using P. guayaquilensis Leaves

1. Leaf Collection
   1. Collect fresh leaves of P. guayaquilensis during the dry season in Guayaquil, Guayas Province, Ecuador (2°10′S, 80°0′W).
      ​NOTE: Voucher specimens of this endemic species were deposited at the GUAY Herbarium, Faculty of Natural Sciences, University of Guayaquil, Ecuador, and at the New York Botanical Garden (NYBG) STEERE HERBARIUM, New York, USA. Additional information is available at: http://sweetgum.nybg.org/science/vh/specimen-details/?irn=2602207
2. Preparation of Leaf Extract
   1. Wash the fresh plant material thoroughly with distilled water.
      NOTE: Ensure complete removal of surface impurities.
   2. Dry the leaves in a tray dryer with forced airflow at 40°C.
      NOTE: Low drying temperatures minimize degradation of thermolabile organic compounds involved in Ag⁺ reduction.
   3. Grind the dried material using a hand mill to obtain a fine powder.
   4. Suspend 10 g of powdered material in 200 mL of distilled water.
   5. Heat the suspension at 70°C under continuous stirring for 1 h.
   6. Allow the extract to cool to room temperature (RT, ~28°C).
   7. Filter the extract under vacuum through qualitative filter paper (Grade 1, pore size 11 µm, diameter 110 mm).
      NOTE: Remove all solid residues from the plant material.
   8. Store the filtered extract at 4°C in amber bottles until further use.
3. Fabrication of Phytogenic AgNPs Using Leaf Extracts
   NOTE: During optimization experiments, vary one parameter per assay while maintaining the remaining parameters constant. Evaluate silver nitrate (AgNO₃) concentration (1–5 mM), extract fraction (10%–50% v/v), reaction time (0.5–1 h), and temperature (25°C–80°C). Optimize synthesis parameters using response surface methodology based on a central composite DoE. Study each factor at five levels (-α, -1, 0, +1, and +α). Use the wavelength of the surface plasmon resonance (SPR) peak (λ_SPR), which correlates with NP size, as the response variable²⁴. The experimental design consists of 16 factorial points, 8 axial points, and 5 center-point replicates, resulting in 29 total experiments.
   1. Prepare AgNO₃ solutions by dissolving the appropriate mass of AgNO₃ in distilled water to obtain concentrations between 1 and 5 mM.
      ​CAUTION: AgNO₃ is corrosive and a strong oxidizing agent that may cause skin and eye irritation. Wear appropriate personal protective equipment, including gloves and eye protection. Avoid exposure to light and dispose of Ag-containing waste according to institutional safety guidelines.
   2. Mix the AgNO₃ solution with the plant extract to obtain extract fractions between 10% and 50% (v/v).
   3. Heat and stir the reaction mixture at the selected temperature (25°C–80°C) for the desired reaction time (0.5–1 h).
   4. Allow the reaction mixture to stand at RT for 24 h.
   5. Centrifuge the brown reaction mixture at 1660 × g for 15 min at RT. Discard the supernatant and collect the solid fraction.
   6. Dry the recovered NPs in an oven at 60°C.

2. Synthesis of AgNPs Using A. spicifera Algal Extracts

1. Algal Collection
   1. Collect A. spicifera algae in Valdivia, Santa Elena Province, Ecuador (1°57′23.91′′S, 80°43′51.33′′W).
2. Preparation of Algal Extract
   1. Prepare the algal extract by following the procedure described in Step 1.2. Replace the plant leaves with A. spicifera algal biomass.
3. Fabrication of Phycogenic AgNPs Using Algal Extracts
   ​NOTE: Perform a series of 12 experiments by varying one parameter at a time while maintaining the remaining parameters constant. Evaluate AgNO₃ concentration (1–5 mM), extract fraction (5%–25% v/v), reaction time (1–4 h), and temperature (25°C–70°C).
   1. Prepare the AgNO₃ solutions and algal extract as described in Steps 1.3.1 and 2.2.
   2. Mix the AgNO₃ solution with the algal extract to obtain an extract fraction of 5%–25% (v/v). Heat and stir the reaction mixture at the selected temperature (25°C–70°C) for the desired reaction time (1–4 h).
   3. Allow the solution to cool to RT for 24 h. Collect the solid fraction after centrifugation and dry it in an oven as described in Steps 1.3.5 and 1.3.6.

3. Synthesis of AgNPs Using Earliella sp.

1. Culture Collection and Maintenance
   1. Transfer actively growing mycelial fragments of Earliella sp. onto Potato Dextrose Agar plates.
      NOTE: The fungal strain used in this study was isolated from environmental samples collected on the campus of the ESPOL, Ecuador. The isolate was taxonomically identified based on morphological characteristics and preserved in the culture collection of the Biotechnology Research Center of Ecuador (CIBE, ESPOL) under the internal code E2. As this strain is maintained in an institutional collection and has not been deposited in an international repository, no RRID or external accession number is available.
   2. Incubate the plates at 28°C for 48 h.
   3. Transfer a mycelial fragment into Potato Dextrose Broth and incubate the culture under constant agitation at 150 rpm for 5 days.
      NOTE: This step promotes biomass production and secretion of extracellular metabolites.
   4. Filter the fungal culture through sterile filter paper to separate the mycelial biomass from the extracellular broth. Collect the filtrate for subsequent NP synthesis.
      NOTE: The extracellular broth contains biomolecules that function as reducing and stabilizing agents during AgNP synthesis.
2. Fabrication of Mycogenic AgNPs
   NOTE: Generate 14 experimental conditions by varying one parameter per assay while maintaining constant volume ratios between the fungal broth and AgNO₃ solution. Evaluate AgNO₃ concentration (1–10 mM), pH (4–10 adjusted using HCl or NaOH), reaction time (1–4 h), and temperature (25°C–70°C).
   ​CAUTION: Hydrochloric acid (HCl) and sodium hydroxide (NaOH) are highly corrosive. Wear appropriate personal protective equipment and perform pH adjustments slowly under continuous stirring to avoid splashing and localized overheating. Use 1 M acid or base solutions during pH adjustment.
   1. Mix the fungal extracellular broth with an AgNO₃ solution at the desired concentration (1–10 mM) using a volume ratio between 2.5% and 10% (v/v) to obtain a final reaction volume of 50 mL. Adjust the final reaction volume according to the experimental condition being tested.
   2. Stir the reaction mixture at the selected temperature (25°C–70°C) for the desired reaction time (1–4 h). Allow the solution to cool to RT for 24 h. Collect the solid fraction after centrifugation and dry it in an oven as described in Steps 1.3.5 and 1.3.6.

4. Synthesis of AuNPs Using C. metallidurans and S. oneidensis

1. Culture Collection and Maintenance
   1. Inoculate frozen cell stocks preserved in 20% glycerol at −80°C of either C. metallidurans CH34 or S. oneidensis MR-1 onto tryptic soy agar plates under aerobic conditions. Incubate the plates at 28°C for 24 h.
   2. Select an individual colony from each plate and inoculate it into 250 mL of tryptic soy broth under aerobic conditions. Incubate the culture at 28°C in a rotary shaker operating at 120 rpm.
   3. Harvest the cells at the stationary phase after 72 h of incubation, when the optical density at 610 nm (OD₆₁₀) reaches approximately 1. Centrifuge the cultures at 10000 × g for 10 min at ~20°C.
   4. Wash the collected pellets twice with sterile 0.9% (w/v) NaCl solution. Concentrate the washed cells to an OD₆₁₀ value of approximately 7.
   5. Generate anoxic concentrated cell stocks by degassing the suspensions and filling the headspace with N₂ gas.
2. Fabrication of Bacteriogenic AuNPs
   NOTE: Use a fractional-factorial matrix to evaluate the influence of environmental factors on AuNP synthesis²⁵. Construct the matrix using 7 factors at 2 levels selected according to previous studies^26,27. Arrange the experiments in an orthogonal matrix and randomize the experimental order to minimize trial-combination bias. The resulting experimental order is presented in Table 1.
   1. Dissolve gold chloride trihydrate (HAuCl₄) in deionized water to prepare a stock solution at 5 g L⁻¹.
      CAUTION: HAuCl₄ is toxic and a strong oxidizing agent. Handle all solutions inside a certified chemical fume hood while wearing appropriate personal protective equipment, including a lab coat, gloves, and safety goggles. Avoid contact with organic materials and dispose of waste according to institutional hazardous waste protocols.
   2. Prepare the experimental reactors by diluting the stock solution to the target concentration in 0.9% (w/v) NaCl solution and adjusting the pH according to the experimental conditions listed in Table 1.
   3. Dispense 50 mL of the prepared metal solution into clean 120 mL serum bottles. Immediately seal each bottle using a butyl rubber stopper.
   4. Secure the stopper using an aluminum crimp seal and a handheld crimper. Sterilize the sealed serum bottles by autoclaving at 121°C for 21 min.
   5. Allow the bottles to cool to RT and visually inspect each bottle for precipitation.
      ​NOTE: Discard any bottles showing visible precipitation and prepare replacement reactors.
   6. Establishment of Anoxic Conditions
      1. Connect each sterilized serum bottle to a gas manifold system equipped with a high-purity nitrogen (N₂) source and a vacuum pump.
      2. Generate an anoxic headspace by performing 21 consecutive purge cycles for each reactor requiring anoxic conditions according to Table 1. During each cycle, apply vacuum followed by nitrogen backfilling for approximately 30 s until the internal pressure returns to approximately atmospheric pressure (~1 atm).
         NOTE: Direct verification of anoxic conditions is not required; however, headspace gas analysis, such as gas chromatography, may be used to confirm oxygen removal when necessary.
         ​CAUTION: Nitrogen gas may displace oxygen and create an asphyxiation hazard in enclosed spaces. Ensure adequate laboratory ventilation and verify secure gas-line connections before use.
      3. Maintain the reactors under slight positive N₂ pressure after the final purge cycle.
      4. Label the reactors from 1–8 according to the experimental order. For example, label the fifth experimental condition as R5.
   7. Addition of Electron Donors and Inoculation
      1. Inject the required volume of sterile anoxic liquid electron donor solution, such as sodium lactate, into the liquid phase of the designated reactors according to Table 1 using a sterile syringe and needle.
      2. Inject the required volume of gaseous electron donor, such as H₂, into the designated reactors headspace using a gas-tight syringe.
      3. Inoculate all biotic reactors by aseptically injecting the required volume of concentrated anoxic microbial stock culture into the liquid phase.
         ​NOTE: Store concentrated stock cultures at 4°C before inoculation to maintain cell viability.
   8. Set-Up of Control Experiments
      1. Prepare an abiotic control by following all steps described in Step 4.2 while replacing the microbial inoculum with an equivalent volume of sterile anoxic culture medium or buffer.
      2. Prepare a killed-cell control by following all steps described in Step 4.2 using an inoculum sterilized by autoclaving.
         ​NOTE: Use the controls to account for abiotic metal reduction and biosorption. Prepare the killed-cell control by autoclaving viable cell stocks at 121°C for 21 min to evaluate the contribution of active microbial mechanisms.
   9. Incubation and Reaction Time
      1. Place all serum bottles in temperature-controlled incubators.
      2. Adjust the incubation temperature according to the experimental conditions specified in Table 1.
      3. Place the incubators on rotary shaker platforms and incubate the reactors at 120 rpm.
      4. Allow the reactions to proceed for 72 h.
         NOTE: Pause the experiment after incubation and store the serum bottles at 4°C until sample processing.
         NOTE: Optionally quantify residual dissolved metal after NP biosynthesis using inductively coupled plasma optical emission spectroscopy to evaluate metal removal efficiency and distinguish NP formation from residual dissolved species.
3. Replication of Optimal Conditions
   1. Identify the reactor condition associated with the maximum metal removal following primary screening and data analysis.
   2. Replicate the selected optimal condition in three independent reactor bottles to confirm reproducibility.
   3. Perform the preparation, reactor set-up, and incubation procedures for the triplicate verification experiments according to Steps 4.1 and 4.2.
      NOTE: After completion of the experiments, temporarily store and label all dissolved solutions as aqueous waste until disposal by certified waste-management companies according to the national Organic Code of the Environment and institutional technical guidelines.

Reactor / Bacteria	Temperature (°C)	Atmosphere	Electron donor	Optical density (OD610)	pH	[Gold] (mM)
1 / CMC	37	N2	H2	0.5	1	2
2 / CMC	28	N2	C3H5O3−	1	1	0.2
3 / SOM	37	O2	C3H5O3−	0.5	1	0.2
4 / CMC	28	O2	H2	1	1	2
5 / SOM	37	N2	H2	1	5	0.2
6 / CMC	37	O2	C3H5O3−	1	5	2
7 / SOM	28	N2	C3H5O3−	0.5	5	2
8 / CMC	28	O2	H2	0.5	5	0.2

Table 1: Fractional factorial experimental design used for bacteriogenic AuNP synthesis. This table summarizes the experimental conditions evaluated for AuNP synthesis using S. oneidensis MR-1 (SOM) or C. metallidurans CH34 (CMC). Variables included incubation temperature, headspace atmosphere, electron donor, inoculum optical density (OD₆₁₀), solution pH, and initial Au³⁺ concentration.

5. Characterization of AgNPs and AuNPs

NOTE: Use ultraviolet–visible (UV–Vis) spectroscopy as an accessible method to confirm NP formation through surface plasmon resonance. Use transmission electron microscopy (TEM) to directly evaluate NP morphology and size. Additional physicochemical characterization may be performed using complementary techniques such as dynamic light scattering (DLS), X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and energy-dispersive X-ray spectroscopy (EDS) to evaluate size distribution, crystallinity, surface chemistry, and elemental composition when more comprehensive characterization is required^28,29.

1. UV–Vis Spectroscopy
   1. Instrument Set-Up
      1. Turn on the UV–Vis spectrophotometer and allow the instrument to warm up for at least 15 min to ensure signal stability.
      2. Select spectral scanning mode and configure the wavelength range from 300 to 800 nm using a data interval of 1 nm.
2. Sample Preparation
   1. Collect aliquots of freshly synthesized AgNP or AuNP suspensions. Dilute the samples with distilled water when necessary to maintain absorbance values within the linear detection range.
3. UV–Vis Measurement of NPs
   1. Perform baseline correction using distilled water as the blank solution.
   2. Transfer the prepared sample into a cuvette, place the cuvette in the sample holder, and record the UV–Vis spectrum over the 300–800 nm range.
   3. Identify the SPR band corresponding to NP formation.
      NOTE: AgNPs typically exhibit SPR bands between 405 and 425 nm, whereas AuNPs generally exhibit SPR bands between 520 and 550 nm.
4. Transmission Electron Microscopy
   ​NOTE: Analyze NP morphology and size using TEM operated at 80 kV and equipped with a high-resolution digital camera.
   1. Transfer 1.5 mL of NP suspension into a centrifuge tube and centrifuge at 5000 × g for 5 min at RT.
   2. Discard the supernatant and retain the pellet.
   3. Add 1 mL of fixation solution containing 4% paraformaldehyde and 5% glutaraldehyde prepared in 0.1 M cacodylate buffer to the pellet. Incubate the sample for 25 h at RT. Then, wash the pellet overnight with 0.1 M sodium cacodylate buffer.
   4. Centrifuge the sample at 5000 × g for 5 min and discard the supernatant.
   5. Add 1 mL of 1% (w/v) osmium tetroxide (OsO₄) prepared in 0.1 M sodium cacodylate buffer for fixation for 3 h.
      ​CAUTION: OsO₄ is highly toxic and volatile. Handle all solutions exclusively inside a certified chemical fume hood while wearing appropriate personal protective equipment, including a lab coat, gloves, and safety goggles.
   6. Centrifuge the sample at 5000 × g for 5 min and discard the supernatant.
   7. Dehydrate the sample using a graded ethanol series. Add 50% ethanol, incubate for 15 min. Repeat the procedure sequentially using 70% and 90%. Complete dehydration with three consecutive incubations in absolute ethanol for 30 min per incubation.
   8. Embed the pellet in epoxy resin at RT according to the manufacturer’s instructions.
   9. Prepare semi-thin sections (~1 µm) and stain the sections with toluidine blue for preliminary observation.
   10. Cut ultrathin sections (~60 nm) using a diamond knife and stain the sections with uranyl acetate followed by lead citrate.
   11. Mount the sections on TEM grids and load them into the transmission electron microscope.
   12. Acquire TEM images to evaluate NP morphology and size.

Results

AgNPs were successfully synthesized through green and biosynthetic routes using different biological reducing agents, and ideal synthesis conditions were identified for each method. In the green synthesis mediated by P. guayaquilensis leaf extract, the optimal conditions consisted of an AgNO₃ concentration of 5 mM, an extract-to-metal solution ratio of 25% (v/v), a reaction temperature of 80°C, and an agitation time of 60 min followed by a resting period of 24 h. Under these conditions, UV–Vis spectro...

Discussion

When comparing the evaluated synthesis routes in terms of feasibility, NP size control, operational cost, reproducibility, and overall process efficiency, plant-mediated synthesis using P. guayaquilensis leaf extract demonstrated the most favorable balance among the AgNP synthesis methods investigated. This protocol provided improved control over NP size through systematic optimization and direct microscopic characterization while operating under mild conditions without prolonged biological cultivation. In addit...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Absolute ethanol	Merck	E7023	TEM dehydration series
Aluminum crimp seals	Dwk Wheaton	224178-01	For serum bottles
Amber glass bottles	Duran	2180154	Light-protected storage
ATR-FTIR spectrometer	Thermo Scientific	Nicolet iS10	Spectral range: 300–3600 cm-1
Autoclave	Yamato	SM300	Sterilization at 121 °C
Butyl rubber stoppers	Dwk Kimble	73827-21	Gas-tight sealing
Centrifuge	BIOBASE	BKC-TH20RL	Bench-top centrifuge
Chloroauric acid trihydrate (HAuCl4·3H2O)	Sigma-Aldrich	27988-77-8	Gold precursor
Diamond knife	Aurion	Dyatom 45	Ultrathin TEM sectioning
Distilled or deionized water	Methrom	N/A	Solution preparation
Epoxy resin (EMBED 812/Epon)	Aurion	EMBED 812	TEM embedding medium
Filter paper, qualitative Grade 1	Merck Whatman	WHA1001110	Pore size: 11 µm; diameter: 110 mm
Fourier transform infrared spectrometer (FTIR)	Thermo Scientific	Nicolet iS10	Spectral range: 300–3600 cm-1
Gas manifold system	Custom-built	N/A	For purge cycles
Gas-tight syringe	Hamilton	1001 RN	1 mL
Glutaraldehyde	VWR	219859510	5% Glutaraldehyde, 4% paraformaldehyde in 0.1 M Sodium Cacodylate Buffer
Glycerol	Sigma-Aldrich	G5516	20% stock preservation
Handheld crimper	Biomed	USYGQ-20Z	Crimp sealing of serum bottles
Hand mill/grinder	Corona	L10000	Plant biomass grinding
Hydrochloric acid (HCl)	Merck	320331	pH adjustment
Hydrogen (H2) gas	Linde	Industrial grade	Electron donor gas
ICP-OES system	Varian	Vista-MPX	Metal quantification
Incubator	Memmert	SM 400	Temperature-controlled incubation
Laboratory oven	Barnstead	M100	Drying nanoparticles
Lead citrate	Merck	7398	TEM staining
Nitric acid (HNO3)	Chem-Lab	CL00.0125	Acid reagent
Nitrogen (N2) gas	Linde	Industrial grade	Anoxic conditions
Osmium tetroxide (OsO4)	Merck	201030	TEM fixation
Paraformaldehyde	VWR	101176-014	5% Glutaraldehyde, 4% paraformaldehyde in 0.1 M Sodium Cacodylate Buffer
Potato Dextrose Agar (PDA)	Merck	110130	Fungal cultivation
Potato Dextrose Broth (PDB)	Merck	P6685	Microbiological medium
Rotary shaker	JoanLab	OS-20Pro	Incubation at controlled agitation
Serum bottles (120 mL)	Wheaton	223748	Borosilicate glass
Silver nitrate (AgNO3)	Sigma-Aldrich	209139	ACS reagent, ≥99.0%
Sodium cacodylate buffer	VWR	11650	5% Glutaraldehyde, 4% paraformaldehyde in 0.1 M Sodium Cacodylate Buffer
Sodium chloride (NaCl)	Carl Roth	3957.1	0.9% washing solution
Sodium hydroxide (NaOH)	Sigma-Aldrich	S5881	pH adjustment
Sodium lactate	Sigma-Aldrich	L7022	Electron donor/carbon source
Sterile syringes and needles	BD	Various	Sterile liquid handling
Stirring hot plate	Biologix	01-310X	Heating and agitation
Toluidine blue	EMS	DcU-10	Semi-thin section staining
Transmission electron microscope (TEM)	FEI	Tecnai Spirit Twin G20	Equipped with high-resolution digital camera
Tray dryer with forced airflow	Handmade	N/A	Biomass drying at 40 °C
Tryptic Soy Agar (TSA)	Oxoid	CM01301	Microbiological medium
Tryptic Soy Broth (TSB)	Carl Roth	X938.1	Microbiological medium
Uranyl acetate	Sigma-Aldrich	94260 Fluka	TEM staining
UV–Vis spectrophotometer	Thermo Scientific	Genesys 10S	UV–Vis spectral analysis
Vacuum pump	KNF or equivalent	N 820 G	For anoxic conditions
Biological Materials
Biological Material	Source	Identifier	Comments/Description
Acanthophora spicifera	Local collection	N/A	Marine algal biomass
Cupriavidus metallidurans CH34	SCK-CEN	LMG 1195	Bacterial strain
Earliella scabrosa	CIBE-ESPOL	E2	Institutional fungal collection
Psidium guayaquilensis	Local collection	N/A	Wild plant biomass
Shewanella oneidensis MR-1	BCCM/LMG	LMG 19005	Culture collection strain