Diamond & Related Materials 141 (2024) 110545

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Diamond & Related Materials

journal homepage: www.elsevier.com/locate/diamond

Antibacterial properties of metal nanoparticles–incorporated activated

carbon composites produced from waste biomass precursor
Marieta Belcheva a, Georgi Georgiev b, Boyko Tsyntsarski b, *, Urszula Szeluga c,
Lyudmila Kabaivanova a, *
a
Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev str. 26, Sofia 1113, Bulgaria
b
Institute of Organic chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev str. 9, Sofia 1113, Bulgaria
c
Centre of Polymer and Carbon Materials, Polish Academy of Sciences, M. Curie-Skłodowskiej Str., 34, 41-819 Zabrze, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Activated carbon-based composite materials, together with the well known antibacterial activity of metal
Antibacterial properties nanoparticles, urged the performance of this study to prepare new antibacterial materials with antimicrobial
Activated carbon activity, as an important step in fighting pathogenic organisms. Activated carbon derived from a waste source -
Metal nanoparticles
almond shells - were combined with metal (Ag, Cu, Mg) nanoparticles, in order to obtain metal composite with
Hydro-pyrolysis
desirable characteristics. The composites were obtained using one-step high-temperature hydro-pyrolysis and
further impregnation of the metal from water solution. Investigation of the structure, chemical composition and
morphology of obtained composites was carried out by scanning electron microscopy, XRD, XPS, elemental and
BET analysis, before testing their antimicrobial activity. Strongest antibacterial effect against E. coli was observed
when 10 % Cu Activated Carbon Composite (ACCCu) was used attaining 100 % reduction of microbial count
even at the starting point. ACCAg, ACCMg and activated carbon only, demonstrated such effect after 24 h. Best
results after treatment of S. aureus were achieved with ACCAg and ACCCu after 24 h.The results indicate that the
antibacterial activity depends on the contact time, bacterial species, nature of the metal and metal concentration.
The novel metal nanoparticles-incorporated activated carbon composites demonstrated very good antibacterial
activity. The investigation provides novel materials with antibacterial properties for further development and
potential application in hygiene devices.

1. Introduction distinguished by high thermal and chemical stability. The production of

Nowadays, there is constant increase of the agents threatening
human health, starting with environmental pollution with chemical and
biological agents, going through COVID-19 pandemic, and ending with
terroristic attacks worldwide [1].
Therefore there appears a strive to develop such methods and ma­
terials to deal with this wide range of hazards in waters, air, soil and air
in public buildings, hospitals and private homes.
Activated carbon (AC) is extraordinary material with high specific
surface area and unique characteristics. It contains micro-, meso- and
macro-pores with dimensions comparable to the size of atoms, mole­
cules, ions, viruses and bacteria. These void spaces of zero electron
density induce intense van der Waals forces from the near proximity of
carbon atoms and they are responsible for adsorption processes. Acti­
vated carbon often have high degree of regeneration, and they are
AC has several advantages, for example the opportunity to use different
waste materials (polymers, biomass, industrial waste, etc.) as a pre­
cursors for their synthesis [2]. The exceptional properties of activated
carbons determine their various applications in different areas. Thus,
they are excellent and versatile adsorbents, effectively adsorbing atoms,
molecules, ions, viruses and bacteria, from water, air and soil [3]. They
have wide range of important applications: adsorptive removal of colors,
odors, and tastes, and other undesirable organic and inorganic pollut­
ants, from drinking waters, and during treatment of industrial waste
waters [4,5]; air purification in inhabited and industrial spaces, such as
restaurants, food processing, and chemical industries; for the purifica­
tion of many chemical, food, and pharmaceutical products; in respira­
tors for work under hostile environments; a variety of gas-phase
applications, such as catalysis, gas and air purification, removal of gases,
trapping chemical vapors, reduction of volatile organic compounds and

* Corresponding authors.
E-mail addresses: Boyko.Tsyntsarski@orgchm.bas.bg (B. Tsyntsarski), lkabaivanova@yahoo.com (L. Kabaivanova).

https://doi.org/10.1016/j.diamond.2023.110545
Received 31 July 2023; Received in revised form 10 October 2023; Accepted 22 October 2023
Available online 26 October 2023
0925-9635/© 2023 Published by Elsevier B.V.
M. Belcheva et al.
odors from smoke, domestic chemicals, pets and municipal waste
[1,6,7]. Activated carbons also can reduce smell from mold spores and
freshen the air.
Their use in medicine and health applications to combat certain
types of bacterial ailments and for the adsorptive removal of certain
toxins and poisons, even life-threatening intoxication and for the puri­
fication of blood or gastrointestinal decontamination of ingested toxins
is developing fast in the last decades [8,9].
Despite that activated carbons are among the best adsorbents
available in the market, there are still several problems and challenges
related to their production and properties, as they do not reduce pet
dander; cannot trap fine particulate matter, except ultrafine fraction; do
not trap some allergens. Size and thickness determining the AC filter
effectiveness, is not always suitable. Sorption capacity and removal rates
of some heavy metals and bacteria are not very high [10]. ACs can retain
bacteria or viruses, but do not kill them. In this connection, some studies
have demonstrated that the pores of ACs, used to treat drinking water,
are heavily occupied by microbes, while other researchers revealed that
bacteria attached to ACs can be resistant to chlorination, due to for­
mation of biofilms [11,12].
Many attempts have been made worldwide to solve these challenges
by using modified AC-based composite materials [13–16].
The interest in metal nanoparticles and metal oxide nanoparticles as
compounds with antibacterial potential is growing fast [17,18].
It is believed that antimicrobial activity of metals is due to the ability
of metal cations to inhibit enzymes, to cause damage to cell membranes,
and to prevent the absorption of vital trace elements by microbes. In
addition, some metals may exhibit direct cytotoxic activity.
It is established that metal nanoparticles (Ag, Cu, Fe, Zn) exhibit a
broad spectrum of antimicrobial activity against various types of mi­
croorganisms, including fungi and Gram-positive and Gram-negative
bacteria [19]. Silver (Ag), copper (Cu) and other metal nanoparticles
that possess high efficiency and biosafety, are used to obtain potential
medical functional materials [20].
The aim of this study is to investigate the influence of different metal
nanoparticles on the morphology, thermal degradation stability, me­
chanical, chemical, barrier and antibacterial properties of 3 samples of
composite materials containing 10 % of Ag, Cu and Mg, regarding E. coli
and S. aureus, as well as samples of Cu containing composite materials,
differing in their metal content - 2.5 %, 5 %, 7.5 %, and 10 %, regarding
E. coli.
2. Materials and methods
2.1. Synthesis of carbon-metal composites
The precursor/biomass waste - almond shells/is subjected to one-
stage hydropyrolisys (simultaneous caronization and activation) at
700 ◦ C for 1 h at heating rate 5 ◦ C/min, and 10 cm3/min water vapor
flow rate. The resulting product is carefully grounded in a mortar and
fractionated through sieves of different sizes, whereas the desired par­
ticle size of 0.4–1 mm is selected, and the the material is washed with
destilled water and dried for 10 h at 150 ◦ C. Hydro-pyrolysis leads to
formation of carbon material, containing high amount (by number and
volume) of pores in a wide range - micropores (< 2 nm), mesopores
(2–50 nm) and macroprores (> 50 nm). The presence of pores on the
surface of the nanoporous carbon materials determine their excellent
adsorption properties towards small atoms, molecules, ions, bacteria,
etc., in gaseous and liquid environment.
The obtained carbon material is soaked by original, developed by us,
impregnation method for 24 h at 20 ◦ C in different saturated water-
alcohol metal salt solutions of AgNO3, Cu(NO3)2 and Mg(NO3)2,
respectively. The metal content in saturated metal-nitrate solutions is
adjusted so, that the finaql activated carbon composite should contains
metal in different final concentrations - 2.5, 5, 7.5 and 10 mass %. After
evaporation of the solutions, the solid products were heated up to 600 ◦ C
2
Diamond & Related Materials 141 (2024) 110545
in Ar for 2 h, at a heating rate of 5 ◦ C/min.
2.2. Scanning electron microscopy (SEM)
The morphological study of the obtained composites was carried out
by scanning electron microscope FEI Quanta 250 FEG (2016, FEI Com­
pany, Thermo Fisher Scientific, USA, Oregon, 5350 NE Dawson Creek
Dr., Hillsboro), with high vacuum and beam deceleration, using an
accelerating voltage of 15.0 kV. FEI Quanta 250 FEG is a large-scale
scanning electron microscope with BF/DF STEM detector. The equip­
ment has low and extended vacuum mode up to 1000 Pa (ESEM) and
temperature control range − 25 ◦ C to +55 ◦ C.
2.3. Elemental analysis
Vario Macro Cube (2019, Elementar Analyzensysteme GmbH, Ele­
mentar - Straße 1, 63,505 Langenselbold, Germany) equipment is
applied for determination of C, H, N, S. The samples were subjected to
combustion in oxygen atmosphere and the obtained gases N2, CO2, H2O,
and SO2 were detected by sensitive thermal conductivity detector in the
same order. Oxygen content was determined by the difference. Vario
Macro Cube works with solid samples (1–50 mg).
2.4. BET analysis
Textural characterization of the samples was performed by using N2
adsorption at − 196 ◦ C on porosimeter apparatus Quantachrome Auto­
sorb iQ-C-XR/MP (2021, Quantachrome, Anton Paar QuantaTec Inc.,
1900 Corporate Dr., Boynton Beach, FL 33426, USA). Prior to the ex­
periments, the samples were outgassed under secondary vacuum at
300 ◦ C overnight. The N2 adsorption isotherms were used to calculate
the specific surface area, SBET and pore volumes.
2.5. X-ray diffraction
Structural characterization of the materials was carried out by
Bruker D8 Advance diffractometer (2008, Östliche Rheinbrückenstraße
49, 76,187 Karlsruhe, Deutschland) with CuKa radiation and LynxEye
detector. The average crystallite size was calculated according to the
Scherrer formula.
Powder X-ray diffraction spectra were collected within the 2θ range
of 5–80◦ , with a 0.02◦ step. The spectra were evaluated by Diffracplus
EVA and Topas 4.2 software.
2.6. XPS experiments
The XPS (X-ray photoelectron spectroscopy) studies were performed
on VG Escalab Mk II spectroscope (2010, Thermo Fisher Scientific, 168
Third Avenue, Waltham, MA, USA) with Al Kα1,2 radiation (hν = 1486.6
eV) and total instrumental resolution of ~1 eV. The pressure in the
chamber is 10− 8 Pa. The C1s line at 285.0 eV is applied as internal
standard for calibration of the binding energy. The element concentra­
tions were evaluated from the integrated peak areas, after Shirley
background subtraction, using theoretical Scofield photoionization
cross-sections.
2.7. Antibacterial properties
The antimicrobial activity of the composites was evaluated against
Gram negative E. coli K-12 (ATCC 25922) and Gram positive S. aureus as
test microorganisms. S. aureus was a clinical strain isolated from throat
swab of a patient at microbiological laboratory of University hospital
“Sofiamed”, Sofia, Bulgaria. The patient's material was processed ac­
cording to good laboratory practice and working protocol of the labo­
ratory. Microbiological identification was based on appearance of
colonies on blood agar (bioMérieux), catalase positive reaction and API
M. Belcheva et al.
Staph test (bioMérieux). The strain was tested for antibiotic (Becton
Dickinson, BD BBL™) susceptibility to Clindamycin (S), Ciprofloxacin
(I), Erythromycin (S), Cefoxitin (S), Gentamicin (S), Penicillin (S), Sul­
famethoxazole/Trimethoprim (S) and Vancomycin (S) according to
“The European Committee on Antimicrobial Susceptibility Testing.
Breakpoint tables for interpretation of MICs and zone diameters. Version
11.0, 2021. http://www.eucast.org”. Pure culture of S.aureus was stored
in eppendorf tube with 10:1 0.9 % NaCl and glycerine at − 20 ◦ C .The
evaluation was performed using slightly modified ASTM Standard Test
Method E 2149–10. Detailed description is given bellow. Preparation of
the bacterial inoculum required to grow a fresh 18 h shake culture of
E. coli and S.aureus, respectively in a sterile nutrient broth (composition
for 1 l: 10 g of tryptone, 5 g of yeast extract and 10 g of sodium chloride).
The colonies were maintained according to good microbiological prac­
tice and examined for purity by creating a streak plate, then used to
prepare a bacterial suspension using a sterile buffer solution (composi­
tion for 1 l: 0.150 g of potassium chloride, 2.25 g of sodium chloride,
0.05 g of sodium bicarbonate, 0.12 g of calcium chloride hexahydrate
and at pH 7.0) until the solution reached 0.5 MF. This solution, with
concentration of 1.5 × 108 colony forming units (CFU/ml), was diluted
with the buffer solution to obtain a final concentration of 1.5 × 106 CFU/
ml and it was the working bacterial dilution. The experiments were
performed in 25 ml sterilised Erlenmeyer flasks. One gram of the com­
posite was maintained in contact with 10 ml of the working bacterial
dilution. After 2 min, 100 μl of the working bacterial dilution was
transferred to a test tube, which was followed by serial dilution and
plating out in duplicate on Petri dishes (10 mm × 90 mm) with nutrient
agar (bioMérieux). The Petri dishes were incubated at 37 ◦ C for 24 h.
These dishes represented the T0 contact time. The flasks were then
placed on a wrist-action shaker for 1 h, 24 h, and 48 h. The bacterial
concentration in the solutions at each fixed time was evaluated by
performing again serial dilutions and standard plate counting tech­
niques. The number of colonies in the Petri dish after incubation was
converted into the number of colonies that form a unit per milliliter
(CFU/ml). The percentage reduction was calculated using the following
formula: % Reduction (CFU/ml) = [(B − A)/B] × 100, where A = CFU/
ml for the flask containing the sample after the specific contact time and
B = CFU/ml for the bacterial control after the same specific contact time
Tx. Two experiments were performed: First experiment is dedicated to
analysis of the composites containing different metal species, particu­
larly Ag, Cu and Mg for both E. coli and S. aureus; Second experiment
represents investigation of the composites containing one metal, but at
different concentrations, particularly 2.5 %, 5 %, 7.5 %, 10 % for E. coli.
For each experiment two controls were prepared - bacterial suspension
/bacteria only/ and bacterial suspension with activated carbon.
3. Results and discussion
3.1. Synthesis methods of composite materials
There are different methods for synthesis of carbon materials. Acti­
vation of carbon materials can be performed by physical and chemical
methods, or a combination of both [21]. Chemical activation is per­
formed by using solid or liquid chemicals such as inorganic acids
(H3PO4), bases (NaOH), carbonates (K2CO3), and salts (ZnCl2); the re­
action mixture is thermally treated for 2 h in a furnace at 300–500 ◦ C
[22].
In addition, various gases can be used for the so called “physical”
activation of the carbon materials. Gaseous oxidizing agents, such as
steam, carbon dioxide, oxygen, or ozone at high temperature
(600–900 ◦ C) react with the carbon via breaking the aromatic structures
in amorphous and crystalline carbon. Water vapor and CO2 are usually
applied, because of their low price and availability. Water vapor is often
preferred as it is more reactive than CO2 at an specific temperature, and
the molecule is smaller which leads to better diffusion, reaction rate and
microporous formation [23]. Water vapor activation leads to a product
3
Diamond & Related Materials 141 (2024) 110545
with increased surface area and porous structure, as well as high sorp­
tion capacity towards different adsorbates [24]. Activation with CO2
leads to more uniform pores, whereas water vapor activation forms a big
variety of micro-, meso- and macropores, which determines wide
application of the obtained carbon product.
Thermochemical treatment can be done also with O2, Cl, H2SO4, air,
etc. Oxidation by oxygen and air is used for modification of the prop­
erties of carbon, and increasing oxygen-containing surface groups,
playing an important role in adsorption process [25,26].
Hydro-pyrolysis (one stage process, involving carbonization and
subsequent activation with water vapor) leads to the formation of a
carbonaceous material containing a large amount (in number and vol­
ume) of pores in a wide range - micropores (< 2 nm), mesopores (2–50
nm) and macropores (>50 nm) [27].
The main methods for metal loading are ion-exchange and impreg­
nataion [28]. Usually impregnantion procedure is faster and leads to
higher metal content, whereas the ion-exchange is slow equilibrium
method. The solid phase synthesis process enables the introduction of
heteroatoms (atoms other than C - S, N, O, metals) into the structure and
volume of carbon composites, while, for example, when using impreg­
nation, ion exchange, spray pyrolysis and other methods, metal particles
and ions are mainly retained on the surface.
The presence of pores on the surface of the nanoporous carbon ma­
terial determines its excellent adsorption properties towards various
sized atoms, molecules, ions, bacteria, viruses, etc., in gas and liquid
media. The localization, coordination and properties of these metal
particles and ions is the key to their antibacterial activity.
Utilization of waste biomass (crushed almond stones) is performed as
follows: the biomass precursor is milled and subjected to high temper­
ature (700 оС for 1 h) hydro‑carbonization, leading to the formation of a
variety of micro-, meso- and macropores, which determines the broad
applications of the obtained carbon products. The metal content in the
resulting carbon composites is around 10 mass %, as detected by XPS
spectroscopy. The obtained carbon-metal composites are subjected to
antimicrobial analysis. After the first promising results, additional car­
bon‑copper composites were perepared, containing 2.5, 5 and 7.5 % Cu,
respectively. The impact of the obtained carbon composites on E. coli
(reference strain) was tested.
3.2. Structural and morphological analysis - SEM
SEM images (Figs. 1-3) of the metal carbon composites show the
presence of metal nano-particles, well embedded in the pores of
Fig. 1. SEM image carbon composite containing Ag.
M. Belcheva et al.
Fig. 2. SEM image of carbon composite containing Cu.
Fig. 3. SEM image of carbon composite containing Mg.
activated carbon, which is well visible in all the samples. ACCAg (Fig. 1)
contains two well distinguished types of uniform ball-like silver nano­
particles, “small balls” nanoparticles (with size around 1 μm) and “large
balls” nanoparticles (around 20 μm), well distinguished on the SEM
image. Copper nanoparticles in ACCCu (Fig. 2) are also uniform ball-like
structures with size around 1 μm. ACCMg (Fig. 3) contains metal
nanoparticles with big variety of sizes and shapes.
3.3. XRD and BET study
XRD data (Fig. 4) show moderate degree of graphitization, which is
proved by the presence of two peaks at 24◦ and 44◦ , respectively, cor­
responding to the (002) and (100) bands of 2H-hexagonal crystalline
graphite. The positions of these two peaks correspond to the average
values of the distances between graphene layers (or between carbon
atoms in directions perpendicular to the sheets) and between carbon
atoms within the layers, respectively. The position and width of the
peaks is related to the mean crystallite size. Our carbon materials are
amorphous on macro scale, however on micro scale they are formed by
4
Diamond & Related Materials 141 (2024) 110545
Fig. 4. XRD spectra of carbon composites.
microcrystalites, which consist of tiny packs of graphene sheets.
XRD results confirm the formation of metal nanoparticles on the
surface of metal‑carbon composites. Ag, Cu2S, CuO and MgO were
detected. In XRD spectrum of ACCCu, Cu nanoparticles were detected at
2θ = 67◦ [29]. There were also Ag containing nanoparticles at 2θ = 32◦
in ACCAg sample [30]. For ACCMg the band at 2θ = 46◦ corresponds to
MgO [31].
N2 adsorption isotherms of all samples are presented in Fig. 5. The
samples are distinguished by high surface area and presence of micro-,
meso- and macropores (Table 1).
All isotherms are according to IUPAC classification [32].
The isotherm presents mixed type, which is hybrid between I and IV
isotherm types, according to the BET classification. A hysteresis loop is
clearly seen in the desorption branch at relative pressures >0:5 – this
could be attributed to capillary condensation of nitrogen in slit-shaped
mesopores [32].
The type and size of metal nanoparticles, as well as porous structure
of carbon (established by BET investigations), determines antibacterial
activity of metal‑carbon composites.
The surface area and pore volume increase after metal addition, due
to penetration of metals, metal oxides and cations in the pores. This
effect increases from silver through magnesium to copper, which is in
agreement with decreasing the size of their atomic and ionic radius.
XPS data (X-ray Photo-electron Spectroscopy, not shown) show the
presence of Ag0, copper in coordinations Cu+ and Cu2+ in ratio 2:1,
Mg2+ (Fig. 6). The content measured by XPS on the surface of the
composites is 12.04 mass. % for Ag, 8.39 mass. % for Cu, and 11.20 at. %
for Mg, respectively.
The equations, which show reduction mechanism of metal nano­
particles, are as follows:
2AgNO3 →2Ag + 2NO2 + O2
2Cu(NO3 )2 →2CuO + 4NO2 + O2
4CuO + C→2Cu2 O + CO2
2 Mg(NO3 )2 →2 MgO + 4 NO2 + O2
Antibacterial properties
In our study we have chosen to combine AC derived from almond
shells with Ag, Cu and Mg-nanoparticles. The resulting composites were
tested against reference strain E.coli K-12 and clinical strain S. aureus.
Working bacterial solution and working bacterial solution with acti­
vated carbon were used as first and second control to ensure that the
microbicidal effect was exclusively due to the composites. Samples were
M. Belcheva et al. Diamond & Related Materials 141 (2024) 110545

a b

c d
Fig. 5. N2 adsorption isotherms at 77 K: ACC (a), ACCMg (b), ACCCu (c), ACCAg (d).

bacteria are most probably in stationary phase during the testing period.
Table 1
Regarding the second control after 1 h exposure the microbial count
Textural parameters of the studied samples.
decreased about 10 times and on the first and the second day no viable
BET surface V total, V micro, V meso, V macro, bacteria in the solution were detected. This might be explained with the
area, m2/g cm3/g cm3/g cm3/g cm3/g
fact that the very characteristics that make activated carbon such an
AC 671 0.350 0.229 0.075 0.055 effective filter also make it hospitable for bacteria. Bacteria easily
ACCCu 595 0.296 0.194 0.054 0.048
adhere to the pores of the activated carbon. When bacteria colonise the
ACCMg 511 0.268 0.175 0.053 0.040
ACCAg 319 0.219 0.100 0.032 0.044
AC surface, they produce a slimy biofilm - an aggregation of microor­
ganisms and extracellular proteins, DNA and sugars secreted from the
cells - that covers the carbon material [33].
taken and plated at different exposure times - 0, 1, 24 and 48 h. The Thus we assume that as time progresses more bacteria leave the so­
obtained carbon-metal composites were subjected to antimicrobial lution and harbor the AC particles and their adhesion to the composites
analysis. Highest antimicrobial activity demonstrated the Cu-containing is a reason for the resulting bacterial reduction. This hypothesis should
composite, and it was selected for further investigation. After the first be further approved for example with death/alive staining and confocal
promising results, additional carbon‑copper composites were prepared, fluorescent microscopy.
containing 2.5 %, 5 % and 7.5 % Cu, respectively and tested against The percentage reduction (% R) of E. coli at different contact time
E. coli. was determined in relation to the bacterial control at Tx contact time,
Fig. 7 is a histogram representing the bacterial concentration of respectively. Data are shown in Table 2.
E. coli (CFU/ml) as a function of the contact time (0, 1, 24, and 48 h). Best antibacterial effect against E. coli was observed when 10 %
Microbial count of the first control remained in the range of 0.8–1.7 × ACCCu was used with 100 % reduction of microbial count at the starting
106, indicating that the cell death was not taking place naturally and point. ACCAg and ACCMg and activated carbon only, showed the same

5
M. Belcheva et al. Diamond & Related Materials 141 (2024) 110545

effect at 24 h. The results indicate that the antibacterial activity depends

on the contact time and the nature of the metal. Our results are similar
and support the results from other studies, where to activated carbon, Ag
[34,35], Cu [36,37] and Mg were added [38].
Similar studies exist on Ag nanoparticles encapsulated into carbon
nanotubes, using silver nitrite (AgNO3) as a precursor and glucose
employed that exhibited an enhanced antibacterial activity against
E. coli, in comparison to Ag nanoparticles supported on activate carbon
prepared by the traditional wetness impregnation method [39]. How­
ever, in our studies 10 % ACCCu, obtained by one step hydropyrolisis
method was used attaining 100 % reduction of microbial count even at
the starting point. ACCAg showed such effect after 24 h. The results
indicate that the antibacterial activity depends on the contact time and
the nature of the metal and metal concentration, as well as on the syn­
thesis method used.
It is not quite possible to compare the data exactly since disk diffu­
a sion method has been applied by some authors, which differs from our
method. Nevertheless, the same trend is established.
In order to evaluate the impact of metal concentration on antibac­
terial effect we proceeded with further testing of the material which
exhibited the best result, namely ACCCu. Taking into consideration the
type and complex structure of the composites studied as an assessment
method instead of minimal inhibitory concentration determination,
changes in the metal concentration within the composite was applied.
Sampling time, controls and bacterial suspension were the same as in the
previously discussed assay with the exception of activated carbon
composite, containing 2.5 %, 5 %, 7.5 %, and 10 % copper were added to
the solution.
Fig. 8 represents the bacterial survival of E. coli (CFU/ml) as a

Table 2
Percentage reduction (% R) of E. coli at different metal (Ag, Cu and Mg) and at
different contact time.
Sample %R (t = 0 h) %R (t = 1 h) %R (t = 24 h) %R (t = 48 h)

b AC 7.4 77.5 100 100

ACCAg 9.9 99.9 100 100
ACCCu 100 100 100 100
Fig. 6. XPS spectra of the studied samples: ACCAg (a) and ACCCu (b).
ACCMg 35.6 87.5 100 100

Fig. 7. Antibacterial effect of ACCAg, ACCCu and ACCMg against E. coli.

6
M. Belcheva et al. Diamond & Related Materials 141 (2024) 110545

Fig. 8. Antibacterial effect of ACCCu at different concentrations against E. coli.

function of the contact time and metal concentration. Addition of Cu to AC gave a wide zone of inhibition against E. coli,
The more the contact time and the higher the copper content, the but not against against S. aureus [36]. In the present research the effect
lower microbial count was obtained. Table 3 gives the reduction (% R) of against S. aureus is stonger. ACCMg also reached 98.6 % reduction but
E. coli in relation to metal concentration at different contact time. Best after 48 h.
antibacterial effect against E.coli with 100 % reduction of microbial Another study [38], where disk diffusion method was used, also
count was observed at starting point when metal concentration of Cu in disloses significant cytotoxic effect of photocatalysts, containing AC and
ACCCu was 7.5 % and above. Activated carbon composites with lower MgO, against both E. coli and S. aureus.
concentration of Cu showed the same effect at 1 h. The higher antibacterial activity against E. coli, as compared to
Another common bacterial species S. aureus came into the scope of S. aureus, might be due to the difference in the cell wall structure of these
our study, since it causes a wide range of infections in all parts of human bacterial species, whereasas the first one belongs to the group of Gram
body but also can asymptomatically colonise skin and nasopharynx. negative bacteria, and the second one to the group of Gram positive
Fig. 9 is a histogram representing the bacterial concentration of S. aureus bacteria. The latter possesses a thick peptidoglycan layer which is un­
(CFU/ml) as a function of the contact time (0, 1, 24, and 48 h). Microbial essential barrier for many harmful chemical compounds.
count of the first and the second control slightly went down from 10− 6 to Metal nanoparticles are well known for their antibacterial activity
10− 4 CFU/ml which is rate of one log per day, most probably beacause and many studies confirm their efficiency as antimicrobial agents, when
the conditions during the testing period did not provide growth opti­ added to wide variety of materials [10]. However, the mechanism of
mum and there was no interaction between S. aureus and the activated antimicrobial activity is not fully clarified. Ag has been known since
carbon. ancient times for its Antimicrobial properties and antibacterial effect of
As described above in previous section as measure of antibacterial silver-based materials have received wide attention due to the low
effect of metal composites a microbial count method was applied. It can toxicity of silver cations towards human cells [41]. By using TEM, it was
be seen on Fig. 10, which is a picture of selected plated Petri dishes after demonstrated that silver nanoparticles can adhere and penetrate into
24 h incubation when the bacterial colonies are visible. Each bacterial E. coli cells, and further they could damage the cell membrane [42]. On
colony counts for one bacterial cell. The lack of colonies on dishes for the other hand, it was reported that Ag+ ions released by silver-based
samples with ACCAg and ACCCu corresponds to their excellent anti­ materials interact with the respiratory chain enzymes of E. coli and
bacterisal potential. inhibit the respiratory chain leading to cell death [43]. Marambio-Jones
The percentage reduction (% R) of S. aureus at different contact time and Hoek [44] suggested that the most common bactericidal mecha­
was determined as for E. coli in relation to the bacterial control at Tx nisms of silver- based materials are as follows: (i) uptake of free silver
contact time, respectively and data are shown in Table 4. Best antibac­ ions followed by disruption of ATP production and DNA replication, (ii)
terial effect against S. aureus was observed when 10 % ACCAg and silver particles and silver ions in the presence of dissolved oxygen
ACCCu were used with 100 % reduction of microbial count at 24 h, generate reactive oxygen species that attack membrane lipids and lead
which is more slower in comparision with E. coli. Another study [40], to a breakdown of membrane and mitochondrial function or cause DNA
which assessed minimum inhibitory concentrations of nanoparticles damage, causing bacterial cell death, and (iii) silver particles directly
consisting of a mixture of silver nitrate and titanium dioxide against damage to the cell membranes. Production of reactive oxygen species
E. coli ATCC 25922 and S. aureus ATCC 25923, also found that S. aureus (ROS) [45] and changes in the cell membranes [46] is probably the
is more tolerant to the nanoparticles. The same was observed in yet bactericidal mechanisms of Mg nanoparticles. There are a lot of re­
another study [35]. searches that reveal strong bactericidal effect of Cu nanoparticles
[47–49]. Cu nanoparticles release ions and ROS, which result in lipid
peroxidation and cause damage to phosphorus- and sulfur-containing
Table 3 biomolecules, which leads to disruption of intracellular biochemical
Percentage reduction (% R) of E. coli at different concentration of Cu and at
processes [50] some studies indicate that the bactericidal properties of
different contact time.
the nanoparticles are size dependent, since the only nanoparticles that
Sample %R (t = 0 h) %R (t = 1 h) %R (t = 24 h) %R (t = 48 h) present a direct interaction with the bacteria preferentially have a
AC 5.7 40 100 100 diameter of ~1–10 nm [51]. Our results show that the investigated
ACCCu (2.5 % 98.3 100 100 100 materials contain Ag, Cu and Mg nanoparticles in the same range ~1–10
Cu)
nm, and presence of Ag0, copper in coordinations Cu+ and Cu2+ in ratio
ACCCu (5.0 % 99.9 100 100 100
Cu)
2:1, and Mg2+. Therefore, we expect that they provide the same mech­
ACCCu (7.5 % 100 100 100 100 anism as discussed above.
Cu)
ACCCu (10 % Cu) 100 100 100 100

7
M. Belcheva et al. Diamond & Related Materials 141 (2024) 110545

Fig. 9. Antibacterial effect of ACCAg, ACCCu and ACCMg against S. aureus.

Fig. 10. Microbial count of S.aureus in bacterial control, control with AC and samples with ACCAg, ACCCu and ACCMg at 1 h and 10,000 x dilution (a), 24 h and 100
x dilution (b), and 48 h and no dilution (c).

8
M. Belcheva et al. Diamond & Related Materials 141 (2024) 110545

Table 4 treatment, Appl Water Sci 10 (2020) 62, https://doi.org/10.1007/s13201-020-

Percentage reduction (% R) of S. aureus at different metal (Ag, Cu and Mg) and at 1145-z.
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