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\DOI{10.5802/crchim.243}
\datereceived{2022-10-24}
\datererevised{2023-05-29}
\daterevised{2023-05-02}
\dateaccepted{2023-07-10}
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%\begin{noXML}

%\RubricEF{Research article}{Article de recherche}

\title{Development of antibacterial magnetic clay-based nanocomposites
for water treatment}

\alttitle{D\'{e}veloppement de nanocomposites antibact\'{e}riens
magn\'{e}tiques \`{a} base d'argile pour le traitement de l'eau}
                                                      
\author{\firstname{Manuel} \lastname{Horue}\CDRorcid{0000-0002-4101-1967}}
\address{Centro de Investigaci\'{o}n y Desarrollo en Fermentaciones Industriales (CINDEFI), 
Laboratorio de Nanobiomateriales, Departamento de Qu\'{i}mica, Facultad de Ciencias Exactas, 
Universidad Nacional de La Plata-CONICET (CCT La Plata), Calle 47 y 115, B1900AJL La Plata, Argentina}

\author{\firstname{Facundo} \lastname{Barraqu\'{e}}\CDRorcid{0000-0002-4683-5527}}
\address{CETMIC, Camino Centenario y 506, B1897ZCA M. B. Gonnet, Argentina}

\author{\firstname{Mar\'{i}a Luciana} \lastname{Montes}\CDRorcid{0000-0001-8702-9719}}
\address{IFLP, Instituto de F\'{i}sica La Plata - CONICET CCT-La Plata, 
Departamento de F\'{i}sica, Facultad de Ciencias Exactas, 
Universidad Nacional de La Plata, Argentina}

\author{\firstname{Mar\'{i}a Emilia} \lastname{Zelaya-Soul\'{e}}\CDRorcid{0000-0002-3737-0035}}
\address{INEDES - CONICET CCT Oca Centenario, Universidad Nacional de Luj\'{a}n, Luj\'{a}n, Argentina}

\author{\firstname{César Fern\'{a}ndez} \lastname{Morantes}}
\address{Grupo de Investigación en Tecnologías y Ambiente (GITA), Corporación Universitaria Autónoma del Cauca, Calle 5 No. 3-85, 190003, Popayán, Colombia}

\author{\firstname{Florencia Camila} \lastname{Urruchua}\CDRorcid{0009-0007-7157-9258}}
\addressSameAs{2}{CETMIC, Camino Centenario y 506, B1897ZCA M. B. Gonnet, Argentina}

\author{\firstname{Hilda Edith} \lastname{Correa}}
\addressSameAs{2}{CETMIC, Camino Centenario y 506, B1897ZCA M. B. Gonnet, Argentina}

\author{\firstname{Guillermo Raul} \lastname{Castro}\CDRorcid{0000-0002-6187-7805}}
\address{Max Planck Laboratory for Structural Biology, 
Chemistry and Molecular Biophysics of Rosario (MPLbioR, UNR-MPIbpC). 
Partner Laboratory of the Max Planck Institute for Biophysical Chemistry (MPIbpC, MPG). 
Centro de Estudios Interdisciplinarios (CEI), Universidad Nacional de Rosario, 
Maip\'{u} 1065, S2000 Rosario, Santa Fe, Argentina}
\address{Nanomedicine Research Unit (Nanomed), Center for Natural and Human Sciences (CCNH), 
Universidade Federal do ABC (UFABC), Santo Andr\'{e}, SP, Brazil}

\author{\firstname{Mariela Alejandra} \lastname{Fernandez}\CDRorcid{0000-0001-6581-1996}\IsCorresp}
\addressSameAs{2}{CETMIC, Camino Centenario y 506, B1897ZCA M. B. Gonnet, Argentina}
\email[M. A. Fernandez]{marielafernandez0712@gmail.com}

\shortrunauthors

\keywords{\kwd{Magnetic-clay composites}
\kwd{Antibacterial materials}
\kwd{Montmorillonite}
\kwd{Organoclay}
\kwd{Surfactant}}

\altkeywords{\kwd{Composites magn\'{e}to-argileux}\kwd{Mat\'{e}riaux
antibact\'{e}riens}\kwd{Montmorillonite}\kwd{Organoargile}\kwd{Tensioactif}}

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\begin{abstract} 
Organoclays were prepared using Argentinean montmorillonite (Mt),
hexadecyltrimethylammonium, or benzalkonium chloride at different
concentrations. Surfactant incorporation, a key factor in biocide
capacity, was estimated using interlayer space and thermogravimetric
analysis (TGA). Subsequently, Fe oxides were grown on the organoclays.
Saturation magnetization indicated that all magnetic composites could
respond to an external magnetic field. Inhibition experiments showed
high antibacterial activity against \textit{Enterococcus faecium},
\textit{Escherichia coli}, and \textit{Salmonella typhimurium}. The
synthesized nanocomposites loaded with 150\% CEC (cation exchange
capacity) of hexadecyltrimethylammonium were the most effective. Those
results were more precisely investigated using the viable plate count
method. The magnetic materials had a similar antimicrobial effect to
those without magnetic properties, indicating that Fe oxides can be
nucleated to impart a magnetic response without affecting the biocide
properties. The organoclays showed higher antibacterial activity
against \textit{E. coli} than against the other bacteria 
assayed. \pagebreak In
addition, the agar disk-diffusion method revealed that antimicrobial
agents were not released from clays, which is a relevant fact in the
water treatment process.
\end{abstract} 

\begin{altabstract}
Des organoargiles ont \'{e}t\'{e} pr\'{e}par\'{e}es \`{a}
partir de montmorillonite argentine (Mt),
d'hexad\'{e}cyltrim\'{e}thylammonium ou de chlorure de benzalkonium
\`{a} diff\'{e}rentes concentrations. L'incorporation de tensioactif,
un facteur cl\'{e} de la capacit\'{e} biocide, a \'{e}t\'{e}
estim\'{e}e en utilisant l'espace entre les couches et l'analyse
thermogravim\'{e}trique (TGA). Par la suite, des oxydes de fer ont
\'{e}t\'{e} form\'{e}s sur les argiles organiques. L'aimantation \`{a}
saturation a indiqu\'{e} que tous les composites magn\'{e}tiques
pouvaient r\'{e}agir \`{a} un champ magn\'{e}tique externe. Les
exp\'{e}riences d'inhibition ont montr\'{e} une forte activit\'{e}
antibact\'{e}rienne contre \textit{Enterococcus faecium},
\textit{Escherichia coli}
et \textit{Salmonella typhimurium}. Les nanocomposites
synth\'{e}tis\'{e}s charg\'{e}s avec 150 \% de CEC (capacit\'{e}
d'\'{e}change cationique) d'hexad\'{e}cyltrim\'{e}thylammonium
\'{e}taient les plus efficaces. Ces r\'{e}sultats ont \'{e}t\'{e}
\'{e}tudi\'{e}s plus pr\'{e}cis\'{e}ment \`{a} l'aide de la m\'{e}thode
de comptage des bact\'{e}ries viables. Les mat\'{e}riaux
magn\'{e}tiques ont eu un effet antimicrobien similaire \`{a} ceux qui
n'avaient pas de propri\'{e}t\'{e}s magn\'{e}tiques, ce qui indique que
les oxydes de fer peuvent \^{e}tre nucl\'{e}\'{e}s pour donner une
r\'{e}ponse magn\'{e}tique sans affecter les propri\'{e}t\'{e}s
biocides. Les organoargiles ont montr\'{e} une activit\'{e}
antibact\'{e}rienne plus \'{e}lev\'{e}e contre \textit{E. coli} que
contre les autres bact\'{e}ries test\'{e}es. En outre, la m\'{e}thode
de diffusion sur disque d'agar a r\'{e}v\'{e}l\'{e} que les agents
antimicrobiens n'\'{e}taient pas relargu\'{e}s par les argiles, ce qui
est un fait pertinent dans le processus de traitement de l'eau.
\end{altabstract}

\dateposted{2024-03-06}
\begin{document}
\maketitle
\twocolumngrid

%\end{noXML}



\section{Introduction}

In different parts of the world, drinking water usually contains a
diversity of pathogens, such as viruses, bacteria, protozoa, and
parasites, which pose a serious risk to human health. In particular,
gram-negative bacteria (\textit{S. typhimurium}, \textit{E. coli},
\textit{Vibrio cholera}, and \textit{Campylobacter jejuni}) and some
gram-positive bacteria (\textit{Bacillus cereus}, and
\textit{Staphylococcus aureus}) have been reported as harmful
microorganisms found in the water of different countries, and the
monitoring and remediation of water containing them is crucial to avoid
associated complications related to the most predominant pathogens
mentioned above  \cite{Naserietal2022}. This is necessary and
encourages the production of simple and economical materials to address
this problem.


Several biocide materials based on clays, such as clays modified by the
sorption of biocide compounds, have been proposed for various
applications, including cosmetics, medicine, food, and the detergent
industry \cite{BuffetBataillonetal2012}. Among biocide compounds,
alkyltrimethylammonium cations, especially quaternary amines, are
commonly employed as organic compounds with antibacterial activity 
\cite{Herreraetal2000,Malachovaetal2009,Ozdemiretal2010,Yarzaetal2020}.
However, the isolated use of these compounds for wastewater treatment
is limited because they are soluble and can become pollutants in the
effluent. Therefore, the employment of adequate support materials that
allow the biocide action of the compounds to avoid their dissolution in
the effluent is a potential alternative.

Clays have been proposed as support materials for biocidal compounds.
Among them, those based on montmorillonite (Mt) and
alkyltrimethylammonium cations have been proposed as antifungal
materials \cite{Karasaetal2014,Yarzaetal2020} and pollutant adsorption
materials \cite{Floresetal2020,Gambaetal2017}. More recently, organoclays with
a magnetic response, that is, organoclays modified by the growth of
magnetic Fe oxide particles, have also been employed for the removal of
heavy metals, dyes, and arsenic
\cite{Barraqueetal2021,Barraqueetal2020,Barraqueetal2018}. The combination of
magnetic nanoparticles with clays has been considered for enzymatic
immobilization \cite{AydemirGuler2015,Zhaoetal2011}, the development of
catalytic materials \cite{Fadillahetal2020,Xuetal2019}, environmental
remediation \cite{Montesetal2020,Wangetal2017,ZelayaSouleetal2021,Zhuetal2021}, and
the design of biocompatible materials \cite{Chenetal2016}. With regard to
water treatment, these materials have the advantage of providing new
sorption sites, but also allowing material recuperation through
magnetic external fields. It avoids direct handling and decreases the
health risk of workers. However, the antibacterial capacity of these
materials and the potential changes caused by Fe growth have not yet
been analyzed. 

The demonstration of antibacterial activity by magnetic organoclays
will allow us to obtain magnetic and biocidal materials in a single
composite. This study aimed to provide an overview of antibacterial
activity against gram-positive and gram-negative bacteria of magnetic
organoclays, synthesized by the incorporation of
hexadecyltrimethylammonium or benzalkonium chloride on an Argentinean
Mt, with posterior Fe oxide nucleation. 


\section{Materials and methods}
\subsection{Materials}

Commercial Argentinean bentonite (Castiglioni Pes  and Co.) from
\textit{R\'{i}o Negro} Province was used as raw  material. The main
bentonite component is montmorillonite (Mt) (88.2\%) with structural
formula
(Si\tsub{3.81}Al\tsub{0.19})(Al\tsub{1.40}Fe\tsup{3+}\tsub{0.27}Mg\tsub{0.32})\!10(OH)\tsub{2}Na\tsub{0.37}Ca\tsub{0.06}{\ubreak}K\tsub{0.02} 
\cite{Yarzaetal2020}. The bentonite has an isoelectric point at pH 2.7,
the external surface area (determined by N\tsub{2} adsorption) is 
66.0~m\tsup{2}/g \cite{ZelayaSouleetal2021}, and its cation exchange
capacity (CEC) of 0.825~mmol/g \cite{Gambaetal2015}. 

The surfactants used for organoclay synthesis were
hexadecyltrimethylammonium bromide (HDTMABr) or benzalkonium chloride
(BAC). HDTMABr (Purity ${>}$ 97\%, MW ${=}$ 364.45~g/mol, and
critical micelle concentrations of 0.9~mM) was provided by Fluka
(Buchs, Switzerland). BAC (95\% purity, 
$\mathrm{MW}_{\mathrm{avg}}$~${=}$ 354~g/mol,
being a mixture of alkylbenzyldimethylammonium chlorides with alkyl
chains of C8, C10, C12, C14, C16, and C18 length) was purchased from
Sigma (Buenos Aires, Argentina).

The KNO\tsub{3} and KOH (99\% purity) used for Fe oxides
synthesis were purchased from Biopack, while Fe
(SO\tsub{4})${\cdot}$7H\tsub{2}O (analytical degree) was
supplied from Cicarelli Lab (Buenos Aires, Argentina).

\textit{Escherichia coli} ATCC 25922, \textit{Enterococcus faecium},
and \textit{Salmonella typhimurium} were donated by the Microbiology
Department, School of Sciences, \textit{Universidad Nacional de} La
Plata (Argentina).

Nutrient broth, nutrient agar, and Mueller Hinton (MH) agar
were purchased from Merck (Darmstadt, Germany), and resazurin from
Sigma (Buenos Aires, Argentina). Ultrapure water (Milli-Q grade, 
$0.22~\upmu\mathrm{m}$ filtered) was employed to prepare all media and
dispersions (Millipore Co., MA, USA).


\subsection{Materials synthesis}

Organoclays were produced in batch conditions by putting in contact
with 15~g of Mt and solutions of HDTMABr or BAC (1~L) with
concentrations equivalent to 50\%, 100\%, or 150\% to the bentonite
CEC. The suspensions were stirred at 400~rpm for 24~h (room
temperature). The obtained products were washed several times with
distilled water, lyophilized, ground, and adequately stored. The
synthesized products were labeled with prefixes H, for bentonite
modified by HDTMABr, (Figure~\ref{fig1}A) or B when BAC
(Figure~\ref{fig1}B) was used, followed by the surfactant concentration
in solution (50, 100, or 150) and finally by Mt from montmorillonite\break
(i.g.: H50Mt).


\begin{figure}[t!]
\includegraphics{fig01}
\caption{\label{fig1}Chemical structures of HDTMABr (A) and BAC (B).}
\end{figure}


Magnetic organoclay was synthesized following a previously reported
procedure \cite{Barraqueetal2020,Bartonkovaetal2007}, using the
organoclays as base materials. For this, 25~mL of 0.3~M
FeSO\tsub{4}${\cdot}$7H\tsub{2}O solution was incorporated into an
organoclay suspension (5.9~g/L water) and kept under continuous shaking
for 2~h. Then 25~mL of 0.49~M KNO\tsub{3} solution and 25~mL of 1.25~M
KOH solution were added, and the suspension was heated to 90~\textdegree
C for 2~min. The system was cooled to room temperature and the obtained
material was washed twice with distilled water. The samples were
recovered by magnetic separation and lyophilized. The materials were
labeled as the organoclays base material but adding Mag at the end of
the name (e.g., H50MtMag). The synthesis was replicated using
raw clay instead of organoclay, and the material was labeled\break MtMag. 

\subsection{Samples characterization}

X-ray diffraction (XRD) patterns were measured using a Philips PW 1710
diffractometer (CuK${\upalpha}$ radiation, 40~kV, 35~mA, 10~s/step,
0.02\textdegree\ step size). To improve the diffraction peak definition
corresponding to the crystallographic plane 001 of Mt, samples were
scanned in oriented mode. For this, samples were previously maintained
at 0.47 relative humidity for 48~h.

Thermogravimetric measurements (TGA) were performed using Rigaku 8121
equipment, with alumina as reference material. For that, 20~mg of
samples were placed on alumina crucibles and heated from 24~\textdegree C
to 1000~\textdegree C, in an air atmosphere, increasing the temperature
at a rate of 10~\textdegree C/min. The TGA first-order derivative (DTGA)
was calculated. The actual surfactant loaded was determined, with
respect to the clay CEC, considering the mass loss between 
150~\textdegree C and 1000~\textdegree C, as previously\break reported
\cite{Yarzaetal2020}. 


The M\"{o}ssbauer spectra of Mag samples were measured at room
temperature employing a spectrometer, in transmission geometry,
containing a \tsup{57}Co/Rh radioactive source (constant acceleration,
512 multichannel detections). A $12~\upmu\mathrm{m}$ thick $\upalpha$-Fe
foil was used for velocity calibration and all the isomer shifts were
referred to this standard. Each M\"{o}ssbauer spectrum was analyzed
using a commercial program that allows the use of a hyperfine magnetic
field and quadrupole splitting distributions
\cite{LagarecRancourt1998}. The total Fe concentration of the samples
was estimated from the M\"{o}ssbauer spectrum, following the procedure
proposed by Montes \cite{Montesetal2016}.

For hysteresis loops measurements, the samples were placed on a
diamagnetic sample holder with a negligible signal. The equipment used
was a LakeShore 7404 vibrating sample magnetometer, applying an
external field between ${\pm}$1.9~T. The extracted magnetic parameters
were the high field susceptibility ($\chi_{\mathrm{hifi}}$), remnant
magnetization (Mr), coercive field (Hc), and saturation magnetization~(Ms).


\subsection{Antimicrobial assays} 
\subsubsection{Microdilution method and minimum\newline inhibitory concentration (MIC)}

Overnight cultures of \textit{E. coli} (ATCC 25922),
\textit{S. typhimurium}, and \textit{E. faecium} were
cultured in fresh nutrient broth from cell stocks preserved at
${-}$80~\textdegree C. After incubation, the bacterial concentration was
adjusted to 1.0 $\times$ 10\tsup{8}~cells/mL to produce the inocula.

The bactericidal activity of Mt, MtMag, H50Mt, H50MtMag, H100Mt,
H100MtMag, H150Mt, H150MtMag, B50Mt, B50MtMag, B100Mt, B100MtMag,
B150Mt, and B150MtMag was evaluated in suspensions of nutrient broth
using the microdilution method in 1.5~mL Eppendorf tubes, according to
International Clinical Standards. The samples were suspended in 
$900~\upmu\mathrm{L}$ of nutrient broth at concentrations of 10.0, 5.0,
2.5, 1.25, and 0.625~g/L, and $100~\upmu\mathrm{L}$ of bacterial
inoculum was added to each tube. The tubes were incubated in a shaker
at 15~rpm at 37~\textdegree C for 24~h. Controls were prepared without
clays or bacteria. The tubes were decanted after incubation, the
supernatants were transferred to a 96-well microtiter plate, and viable
cells were quantified by the reduction of the vital dye resazurin assay
\cite{Elshikhetal2016}. Finally, the MIC was determined for each\break
sample. 

\subsubsection{Agar disk-diffusion method}

The presence of growth inhibition haloes was assayed to evaluate the
extent of biocide activity. The procedure was carried out according to
the guidelines described by the International Clinical Standards
(CLSI/NCCLS) using sterile glass cylinders of 8~mm 6~mm 10~mm (external
and internal diameter, length) containing sample suspensions instead of
paper disks. Samples with a relatively high amount of surfactant
(H150Mt, H150MtMag, B150Mt, and B150MtMag) were considered for this
assay, employing 10~g/L of each suspension, while Mt and MtMag were
used as control samples. Overnight cultures of \textit{E. coli},
\textit{S. typhimurium}, and \textit{E. faecium} were used to prepare
adjusted inocula of 1.5 $\times$ 10\tsup{8}~cells/mL and then employed
as inoculum to seed MH agar plates. The cylinders were placed after
inoculation and  $50~\upmu\mathrm{M}$ of each sample was dropped into
the respective cylinder. Incubation was carried out at 37~\textdegree C
for 24~h.

\subsubsection{Assays in Erlenmeyer flasks}

Assays in Erlenmeyer flasks were performed using only the samples
with the highest antimicrobial efficiency based on the
microdilution method results. The tests were performed in 50-mL
Erlenmeyer flasks, and the viable plate count method was used to
enumerate viable bacteria.

In this procedure, overnight cultures of each strain were employed as
inoculum (1.5 $\times$ 10\tsup{8}~cells/mL). The assayed materials
(H150Mt and H150MtMag) were dispersed in fresh nutrient broth at
different concentrations, depending on the susceptibility of each
bacterium. Specifically, 0.25~g/L was added to fresh media against
\textit{E. coli}, 0.50~g/L against \textit{S. typhimurium}, and
0.80~g/L against \textit{E. faecium}. Bacterial inoculum was added to a
final volume of 20~mL. The microdilution method showed that the MtMag
samples were harmless to all strains. Based on these results, a control
constituted by MtMag was used at the corresponding concentration in all
cases. The incubation conditions were 37~\textdegree C and orbital
shaking at 200~rpm for 24~h. After the incubation period, aliquots were
taken from each sample and dilutions ranging from 10\tsup{-1} to
10\tsup{-9} were prepared. The cells were then plated on nutrient agar
and incubated at 37~\textdegree C for 24~h. Finally, the colony-forming
units (CFU) were quantified. All experiments were performed in
triplicate.

\subsection{Statistical analysis}

Data obtained from the viable plate count method were graphed and
analyzed using GraphPad Prism version 6.00 software (GraphPad). Data
related to the number of viable bacteria were analyzed using Student's
$t$-test. Differences between groups were considered significant
when $p$-values were lower than 0.05 (95\% confidence level).


\begin{table*}[t!]
\caption{\label{tab1}The d001 (nm) and the actual organic load (\% CEC,
calculated from TGA) indicate the organic content in the external
surface (VdW) and the interlayer space (exchanged)\vspace*{-1pt}}
\begin{tabular}{ccccc}
\thead
Sample & d001 (nm) & Actual load (\%~CEC) & VdW (\%~CEC) & Exchanged (\%~CEC) \\
\endthead 
\morerows{1}{Mt*} &  1.26 (Na\tsup{+}) &  \morerows{1}{0} 
&  \morerows{1}{0} & \morerows{1}{0} \\  
& 1.43 (Ca\tsup{2+}) & & &\vspace*{2pt} \\
\morerows{1}{MtMag*} &  1.27 (Na\tsup{+}/Fe)  & 
\morerows{1}{0} &  \morerows{1}{0} & \morerows{1}{0} \\  
& 1.47 (Ca\tsup{2+}) & & &\vspace*{2pt} \\
H50Mt*     &  1.5 &  43  $\pm$ 4  &  23 $\pm$ 2 &    20 $\pm$ 2 \\  
H100Mt*    &  2.1 &  84  $\pm$ 7  &  59 $\pm$ 4 &    25 $\pm$ 2 \\  
H150Mt*    &  2.2 &  116 $\pm$ 10 &  89 $\pm$ 8 &    27 $\pm$ 3 \\  
H50MtMag*  &  1.6 &  36  $\pm$ 3  &  16 $\pm$ 2 &    20 $\pm$ 2 \\  
H100MtMag* &  2.0 &  68  $\pm$ 6  &  38 $\pm$ 3 &    30 $\pm$ 2 \\  
H150MtMag* &  2.2 &  90  $\pm$ 9  &  63 $\pm$ 6 &    27 $\pm$ 3 \\  
B50Mt      &  1.6 &  41  $\pm$ 4  &  14 $\pm$ 1 &    27 $\pm$ 3 \\  
B100Mt     &  1.8 &  72  $\pm$ 7  &  52 $\pm$ 4 &    20 $\pm$ 2 \\  
B150Mt     &  2.0 &  79  $\pm$ 7  &  58 $\pm$ 6 &    21 $\pm$ 2 \\  
B50MtMag   &  1.6 &  30  $\pm$ 3  &  23 $\pm$ 2 &    13 $\pm$ 1 \\  
B100MtMag  &  1.9 &  57  $\pm$ 6  &  36 $\pm$ 3 &    21 $\pm$ 2 \\  
B150MtMag  &  1.9 &  36  $\pm$ 6  &  41 $\pm$ 4 &    25 $\pm$ 2   
\botline
\end{tabular}
\xxxtabnote{Note: Previously reported \cite{Barraqueetal2020}.\vspace*{-3pt}}
\end{table*}


\section{Results}
\subsection{Sample characterization}

Table~\ref{tab1} shows the d001 values (corresponding to the interlayer
space of Mt) for all considered samples. Organic samples presented d001
values higher than those of Mt, which indicated the entrance of the
surfactant into the Mt interlayer space. The d001 values, previously
reported for H50Mt, H100Mt, and H150Mt were 1.5, 2.1, and 2.2~nm,
respectively \cite{Barraqueetal2020}, revealing a monolayer arrangement
for H50Mt but pseudo-trilayer for the others 
\cite{MeleshynBunnenberg2006,Schamperaetal2016}. Similar d001 values
and surfactant arrangements were observed for magnetic samples based on
HDTMA clays, indicating that Fe oxide synthesis does not significantly
modify the size of the interlayer space of the organoclays. The
determined d001 values for B50Mt, B100Mt, and B150Mt were 1.6~nm, 
1.8~nm, and 2.0~nm, respectively, which indicates surfactant
arrangements corresponding with monolayer for B50Mt, bilayer for
B100Mt, and pseudo trilayer for B150Mt 
\cite{MeleshynBunnenberg2006,Schamperaetal2016}. Depending on the layer
charge (${=}$interlayer cation density ${=}$ packing density of the
alkylammonium ions) of the clay mineral and the chain length of the
organic ion, different arrangements of organic molecules between the
layers can be formed. The geometry of the surface and the degree of
exchange can also influence. The organic ions may lie flat on the
silicate surface as a monolayer or bilayer or, depending on the packing
density and the chain length, an inclined pseudo trilayer structure can
be formed, with the chains radiating away from the silicate surface.
For HDTMA-based clays, the magnetic composites attained for BAC
presented similar \textit{d001} values to those of the organoclays,
revealing that more surfactant molecules remained in the interlayer
space after Fe oxide synthesis. 

\begin{table*}[t!]
\caption{\label{tab2}Hyperfine parameters extracted from M\"{o}ssbauer spectra
fittings\vspace*{-2pt}}
\begin{tabular}{cccccccccc}
%\begin{tabular}{ccccccccccc}
\thead
\multicolumn{10}{c}{Paramagnetic sites}  \\
\cline{1-10}
\xmorerows{1}{Sample} & \multicolumn{3}{c}{Fe\tsup{3+}(I)} & 
\multicolumn{3}{c}{Fe\tsup{3+}(II)} & \multicolumn{3}{c}{RP}  \\
\cline{2-4}\cline{5-7}\cline{8-10}
&  $\delta$ & $\Delta$ & RSA & $\delta$ &  $\Delta$ & RSA &
$\delta$ & $\Delta$ & RSA  \\
\endthead
B50MtMag  &  0.33\tsub{0.20} &  0.65\tsub{0.0}  &  10 $\pm$ 2 &  0.39\tsub{0.2}  &  1.88\tsub{0.15} &  1 $\pm$ 1 &  0.45\tsub{0.10} & 0.05\tsub{7.86}  & 33 $\pm$ 5  \\
B100MtMag &  0.30\tsub{0.19} &  0.65\tsub{0.27} &  \09 $\pm$ 1  &  0.35\tsub{0.10} &  1.70\tsub{0.15} &  ${<}$1      &  0.35\tsub{0.1}  & 2.90\tsub{10.66} & 25 $\pm$ 4  \\
B150MtMag &  0.35\tsub{0.08} &  0.50\tsub{0.16} &  \07 $\pm$ 1  &  0.30\tsub{0.20} &  1.15\tsub{0.20} &  3 $\pm$ 1 &  0.41\tsub{3.33} & 0.0\tsub{7.25}   & 21 $\pm$ 4  \\
\end{tabular}
\begin{tabular}{ccccccccccc}\thead
\multicolumn{11}{c}{Magnetic sites} \\
\cline{1-11}
\xmorerows{1}{Sample}  & \multicolumn{3}{c}{Fe\tsub{3}O\tsub{4} (A site)} &
\multicolumn{3}{c}{Fe\tsub{3}O\tsub{4} (B site)} &
\multicolumn{4}{c}{$\upalpha$-FeOOH} \\
\cline{2-4}\cline{5-7}\cline{8-11}
&  $\delta$ & H &  RSA & $\delta$ &  H & RSA &
$\delta$ &  $\varepsilon$ & H & RSA \\
\endthead
B50MtMag  &  0.36\tsub{0.24} &  47.5\tsub{1.0}  &  19 $\pm$ 3 & 0.44\tsub{0.25} &  43.3\tsub{2.2} & 30 $\pm$ 4 & 0.43\tsub{0.15} &  $-$0.14\tsub{0.1} & 35.5\tsub{2.0} & 7 $\pm$ 1 \\
B100MtMag &  0.31\tsub{0.17} &  48.21\tsub{0.8} &  31 $\pm$ 4 & 0.55\tsub{0.18}  &  44.9\tsub{2.3} & 31 $\pm$ 4 & 0.32\tsub{0.00} &  $-$0.14\tsub{0.1} & 36.6\tsub{0.9} & 4 $\pm$ 1 \\
B150MtMag &  0.30\tsub{0.18} &  48.1\tsub{0.31} &  25 $\pm$ 4 & 0.55\tsub{0.28}  &  44.5\tsub{2.1} & 40 $\pm$ 3 & 0.43\tsub{0.10} &  $-$0.14\tsub{0.1} & 35.6\tsub{2.0} & 4 $\pm$ 2 
\botline
\end{tabular}
\tabnote{Symbols: $\delta$, $\Delta$, and
$\varepsilon$ represent the isomer shift, the quadrupole splitting,
and the quadrupole shift, respectively, in mm/s, while H denotes the
hyperfine magnetic field ($T$) and RSA the relative spectral area for
each proposed Fe site.\vspace*{-3pt}}
\end{table*}






The TGA for both surfactants allowed us to observe that the actual
organic content in the samples was lower than the concentration in the
suspension, indicating that not all available surfactants were
incorporated into the Mt surfaces. The mass loss percentage was
determined from TGA data analysis, while the actual surfactant loading
for all exchanged samples was calculated considering the mass loss of
the MtMag structural hydroxyl groups. The actual values of \% CEC of
the organomagnetic samples were lower than the theoretical ones,
calculated from the initial amount of HDTMABr applied. This behavior
has been mainly attributed to two factors: incomplete exchange of
HDTMA+ as observed for the actual CEC obtained for organic Mt, and
increased release of surfactant during the synthesis of magnetic
material, as previously determined~\cite{Barraqueetal2018}.

In addition, a higher amount of surfactant incorporated in the
synthesis implies a higher surfactant content on the clay surface, but
the contents calculated by TGA indicate that there is no saturation of
the clay CEC with the amount of amine incorporated. For example, for
the H50Mt sample, approximately 43\% of amine was incorporated. 

Considering the data of the actual load for organoclays and their
magnetic counterparts, it can be noted that during Fe oxide synthesis,
a minor part of the surfactant is lost, that is, more surfactant
remains in the composites. The actual organic load obtained for both
surfactants at equal concentrations seems to be similar for the two
lower concentrations; however, in the case of 150\% CEC, B150Mt
presented a lower organic incorporation than H150Mt. This can be
related to the structural characteristics of the BAC molecule, which
presents a structure with two phenyl rings, while HDTMA presents a
head-tail structure, with BAC higher than HDTMA. The small size of
HDTMA makes its incorporation easier at relatively higher
concentrations of Mt in BAC molecules. This hypothesis is also
supported by evidence that the surfactant percentage on the interlayer
was higher for HDTMA than for BAC. These differences in the Mt loading
and their distribution could cause differences in the Fe content and
magnetite formation in the composites, as revealed previously for HDTMA
\cite{Barraqueetal2018}. To analyze this issue, magnetic and hyperfine
characterization of BAC samples was performed, as discussed below.

Figure~\ref{fig2} shows the M\"{o}ssbauer spectra and the proposed fit
for the magnetic organoclay obtained using BAC. The spectra for samples
containing HDTMA have already been reported \cite{Barraqueetal2020}. Six Fe
environments were proposed (Table~\ref{tab2}): two paramagnetic
Fe\tsup{3+}(called Fe\tsup{3+}(I) and Fe\tsup{3+}(II)), one associated
with Fe in paramagnetic relaxation (PR), the two expected sites for
magnetite (Fe\tsub{3}O\tsub{4} A and B), and another corresponding to
goethite ($\upalpha$-FeOOH). 

\begin{figure*}[t!]
{\vspace*{2pt}}
\includegraphics{fig02}
{\vspace*{2pt}}
\caption{\label{fig2}M\"{o}ssbauer spectra of magnetic organoclays synthesized
using BAC. Dots represent experimental data, and the red line is the
resultant fit.}
{\vspace*{2pt}}
\end{figure*}

Paramagnetic Fe\tsup{3+} environments and paramagnetic relaxation can
be associated with the presence of Mt \cite{Barraqueetal2018}, while
magnetite and goethite are the Fe oxides synthesized during alkaline
oxidation in the presence of nitrate. The relative spectral area (RSA)
for magnetite was 49 $\pm$ 5\% for B50MtMag, 62 $\pm$ 6\% for
B100MtMag, and 65 $\pm$ 6\% for B150MtMag, while goethite presented
values around 5\% for all the samples. The ideal magnetite reveals a
ratio between the RSA of the B and A sites of 1.8
\cite{VandenbergheDeGrave2013}, allowing us to conclude that the
synthesized magnetite on the organoclays presented different grades of
oxidation, owing to the obtained ratios for B50MtMag, B100MtMag, and
B150MtMag, which were 1.58, 1.00, and 1.60, respectively. Based on
these results and a previous report \cite{VandenbergheDeGrave2013}, the
attained magnetite oxides on BAC samples present structural formulas of
Fe\tsub{2.99}O\tsub{4}, Fe\tsub{2.92}O\tsub{4}, and
Fe\tsub{2.96}O\tsub{4} for B50MtMag, B100MtMag, and B150MtMag,
respectively, and the oxidized form seems to be maghemite, with
hyperfine parameters similar to magnetite and identical diffraction
patterns.

The actual magnetite content was related to the magnetic response and
was estimated by considering the total Fe concentration of each sample
(Table~\ref{tab3}). The total Fe concentration was determined from the
M\"{o}ssbauer spectra using the approximate method described previously
\cite{Montesetal2016}. The Fe concentrations associated with magnetite
(Table~\ref{tab3}) were similar for the three magnetic composites
developed using BAC, whereas the values were lower than those reported
for H50MtMag, H100MtMag, and H150MtMag of 134, 152, and 131~g/kg,
respectively  \cite{Barraqueetal2020}.  In the same work, it was
concluded that the HDTMA molecules located on the internal surface of
Mt would decrease the incorporation of Fe at that site, leaving more Fe
atoms~free for magnetite formation on the external surface of the clay.
The Fe atoms on the inner surface of~the clay do not favor magnetite
formation. According to TGA analysis, the surfactant loading in the\unskip\break
interlayer space is higher with HDTMA than with BAC for the same
initial surfactant concentration. This result may be associated with
the larger volume of BAC molecules than that of HDTMA. Similar
interlayer Fe concentrations were determined for magnetic organoclays
prepared from organoclays with the same initial surfactant
concentration. Larger surfactant molecules could hinder Fe
incorporation into the interlayer space. Thus, despite the lower BAC
concentration in the interlayer space compared with HDTMA, the
incorporation of Fe ions into the Mt interlayer space could be just as
difficult, leaving Fe ions available for oxide formation. For the
BAC-based magnetic compounds, the magnetite content (Table~\ref{tab3})
was similar for all compounds, despite differences in Fe concentration.

\begin{figure*}[t!]
\vspace*{-2pt}
\includegraphics{fig03}
\vspace*{-2pt}
\caption{\label{fig3}Hysteresis loops of magnetic organoclays obtained using BAC
surfactant.}
\end{figure*}

\begin{table*}[t!]
\caption{\label{tab3}Total Fe concentration, Fe in magnetite (Fe\tsub{3}O\tsub{4}),
and magnetic parameters extracted from hysteresis loops: high field
susceptibility, coercive field Hc, remanent magnetization Mr, and
saturation magnetization Ms\vspace*{-2pt}}
\begin{tabular}{cccccccc}
\thead
Sample &  
\parbox[t]{1.5cm}{\centering Total Fe   (g/kg)}  &
\parbox[t]{1cm}{\centering Fe\tsub{3}O\tsub{4} (\%)} &
\parbox[t]{1.8cm}{\centering Fe in Fe\tsub{3}O\tsub{4} (g/kg)} &
\parbox[t]{1.8cm}{\centering $\chi_{\mathrm{hifi}}$ (10\tsup{-7}~m\tsup{3}/kg)} &   
\parbox[t]{1cm}{\centering Hc (mT)} &
\parbox[t]{1.5cm}{\centering Mr (A${\cdot}$m\tsup{2}/kg)} &
\parbox[t]{1.5cm}{\centering Ms (A${\cdot}$m\tsup{2}/kg)}\vspace*{2pt} \\
\endthead
BMt50Mag  & 206 $\pm$ 14 & 49 $\pm$ 5 & 100  $\pm$  10  & 7.8 $\pm$ 0.5 & \02 $\pm$ 2  & 0.40 $\pm$ 0.10 & 13.5 $\pm$ 0.2 \\
BMt100Mag & 172 $\pm$ 20 & 62 $\pm$ 6 & 107  $\pm$  13  & 4.5 $\pm$ 0.3 & \09 $\pm$ 2  & 2.36 $\pm$ 0.05 & 13.9 $\pm$ 0.2 \\
BMt150Mag & 166 $\pm$ 16 & 65 $\pm$ 5 & 108  $\pm$  11  & 5.0 $\pm$ 0.3 & 14 $\pm$ 2 & 3.14 $\pm$ 0.02 & 15.3 $\pm$ 0.2 
\botline
\end{tabular}
\xxtabnote{Note: Results for samples based on HDTMA were previously
reported \cite{Barraqueetal2020}.\vspace*{-2pt}}
\end{table*}

Going deeper into the magnetic characterization, Table~\ref{tab3} also
presents the magnetic parameters extracted from hysteresis loops. The
remnant magnetization and coercive field were determined for all
samples, indicating hysteresis behavior and magnetite particles with
sizes greater than 36~nm. In addition, the high field susceptibility,
also observed for all samples, indicated the presence of particles with
superparamagnetic behavior, that is, particles with sizes lower than
36~nm \cite{Dunlop1973}. This result was observed in Figure~\ref{fig3}.
These observations have been previously
reported for H50MtMag, H100MtMag, and H150MtMag samples, as well as for
magnetite particles formed on beidellite clays  \cite{Montesetal2020}
and a material composed of MtMag-hydrothermal carbon
\cite{Barraqueetal2020,ZelayaSouleetal2021}. Saturation magnetization
\mbox{determined} for BAC samples resulted in 13.5~A${\cdot}$m\tsup{2}/kg
(BMt50Mag),  13.9~A${\cdot}$m\tsup{2}/kg (BMt100Mag), and
15.3~A${\cdot}$m\tsup{2}/kg (BMt150Mag). The higher value determined
for BMt150Mag could be attributed to the fact that the magnetite formed
on B150MtMag was less oxidized than that formed on B100MtMag. The
saturation magnetization values obtained for all BAC-based organoclays
suggest that they can be manipulated by an external magnetic field.

\vspace*{-1pt}

\subsection{Microdilution method and minimum inhibitory concentration (MIC)}

\vspace*{-1pt}


The results of the antimicrobial activity of organoclay against
\textit{E. faecium}, \textit{E. coli}, and \textit{S. typhimurium}
using the microdilution method revealed with resazurin were used to
determine the MIC (Table~\ref{tab4}). Mt and MtMag did not present
bactericidal activity as well as H50Mt, H50MtMag, B50Mt, and B50MtMag
samples, probably because surfactant concentration was lower than the
minimum necessary to have antibacterial activity in these cases. The
aforementioned samples presented relatively low surfactant
concentrations at the external surface (Table~\ref{tab1}), which is
the main factor responsible for the biocide characteristics of the clay
\cite{Yarzaetal2020}. The samples synthesized using surfactant
concentrations equivalent to 100\% CEC revealed  variable behavior,
whereas samples obtained at 150\% CEC presented the highest
bactericidal activity, in agreement with the higher concentration of
surfactant at the external surface of the composites. Specifically,
viable cells could not be detected with the H150Mt and H150MtMag
sorbents at the concentrations assayed for all strains. In addition, it
is important to highlight that H100MtMag, B100MtMag, H150MtMag, and
B150MtMag presented biocide activity similar to that of non-magnetic
variants, which allows the inclusion of iron oxides for manipulation
with sorbent purposes without modifying the bactericidal activity.
Among the microorganisms assayed, \textit{E. coli} seemed to be the
most susceptible to the biocide action of organoclays in all cases.

\begin{table}[t!]
\caption{\label{tab4}Minimum inhibitory concentration (MIC) for different samples
in \textit{E. coli, S. typhimurium}, and \textit{E.
faecium}}
\fontsize{9}{10.8}\selectfont
\begin{tabular}{cccc}
\thead
\textit{E. coli} & \textit{E. coli} & \textit{S. typhimurium} & \textit{E. faecium} \\
Sample & MIC (g/L) & MIC (g/L) & MIC (g/L) \\ 
\endthead
Mt & ND* & ND* & ND* \\ 
MtMag & ND* & ND* & ND* \\ 
H50Mt & ND* & ND* & ND* \\ 
H50MtMag & ND* & ND* & ND* \\ 
H100Mt & 1.25 & 10.0 & 10.0 \\ 
H100MtMag & 1.25 & 10.0 & 5.0 \\ 
H150Mt & ${<}$0.625** & ${<}$0.625** & ${<}$0.625** \\ 
H150MtMag & ${<}$0.625** & ${<}$0.625** & ${<}$0.625** \\ 
B50Mt & ND* & ND* & ND* \\ 
B50MtMag  & ND* & ND* & ND* \\ 
B100Mt & ${<}$0.625** & ND* & 1.25 \\ 
B100MtMag & ${<}$0.625**  & ND* & ND* \\ 
B150Mt & ${<}$0.625** & 1.25  & ${<}$0.625** \\ 
B150MtMag & ${<}$0.625**  & 1.25 & 1.25 
\botline
\end{tabular}
\tabnote{Notes: *ND means non-determinate cause in the experiment the
highest clay concentration (10.0~g/L) could not inhibit bacterial
growth. **${<}$0.625~g/L means that bacterial growth could not be
detected for the lowest concentration assayed in the experiment 
(0.625~g/L).}
\end{table}

In conclusion, non-viable bacteria were detected in H150Mt and
H150MtMag samples at all the concentrations tested. They became the
most efficient system and were selected as the samples for assays in
Erlenmeyer flasks.

\subsection{Agar disk diffusion method}

This method is usually employed to determine the susceptibility of a
bacterial strain to antimicrobial compounds. A clear zone, ring, or
halo without bacterial development represents growth inhibition, which
depends on the antimicrobial effectiveness of the compound, its
concentration, and its ability to diffuse through the agar.
Figure~\ref{fig4} shows bacterial lawns after incubation. It is
possible to appreciate the absence of an inhibition halo in all cases.
According to the microdilution method, the concentrations assayed 
(10~mg/mL) of H150Mt, H150MtMag, B150Mt, and B150MtMag were over the
minimum value necessary to inhibit bacterial growth in liquid media.
Therefore, they must be suspended and kept under constant stirring
during water treatment to ensure intimate contact between them and
bacteria. The fact that surfactants are not released into the water is
an advantage, as if they were, they could be remnant pollutants.

\begin{figure*}[t!]
\includegraphics{fig04}
\caption{\label{fig4}Plates employed in agar-disk diffusion method; samples assayed
were (A--C) H150Mt, H150MtMag; (D--F) B150Mt, H150MtMag; controls assayed
were (A--F) Mt, MtMag.}
\end{figure*}


\subsection{Assays in Erlenmeyer flasks}

The present experiment was carried out with information provided by the
microdilution method and MIC, which was used as a guide to determine
which material concentration must be applied to ensure suitable growth  
inhibition. The results revealed that the most susceptible bacterium
was \textit{E.~coli}; in agreement with the results of the
microdilution method, a concentration of 0.25~mg/mL of material was
required to cause bacterial inhibition in this case 
(Figure~\ref{fig5}). The second one was \textit{S. typhimurium} which
required 0.50~mg/mL of material to be inhibited. The most resistant
bacterial strain was \textit{E. faecium} which required 0.80~mg/mL.
Furthermore, there were no significant differences between H150Mt and
H150MtMag; thus, the growth of Fe oxides did not affect the
antibacterial activity of surfactant-loaded clays. This result
indicates that this modification can be performed without damaging the
biocidal properties of the compounds, with the advantage of providing a
magnetic response that can be used to remove the material relatively
easily without direct contact after the remediation process.

\begin{figure*}[t!]
\includegraphics{fig05}
\vspace*{4pt}
\caption{\label{fig5}The number of viable bacteria for (A) assay against \textit{E.
coli} (material concentration 0.25~mg/mL, (B) assay against \textit{S.
typhimurium} (material concentration 0.50~mg/mL, and (C) assay against
\textit{E. faecium} (material concentration 0.80~mg/mL).}
\vspace*{-6pt}
\end{figure*}

\section{Conclusions} 

Antimicrobial materials for water treatment were synthesized by
modifying Argentine montmorillonite with hexadecyltrimethylammonium or
benzalkonium chloride at different concentrations, with the posterior
growth of Fe oxides. The chosen Fe oxide synthesis was adequate to
impart a magnetic response to the biocide materials; only a small
amount of surfactant was lost during the procedure, without significant
antibacterial activity. The surfactant molecules were adsorbed on the
external and internal surfaces of Mt. From an antimicrobial point of
view, the surfactant located at the external surface is more relevant
than that located at the internal surface; thus, the samples that
showed higher surfactant presence on the external surface were those
that were loaded with the highest surfactant concentrations. The
material developed using the highest concentration of HDTMA had the
highest surfactant load on the external surface, which is probably
related to the smaller size of HDTMA compared to BAC. Magnetite was
the main Fe oxide formed, and the saturation magnetization of all
composites suggests adequate manipulation by an external magnetic
field.

Although the Mt and MtMag samples did not show antimicrobial activity,
the synthesized organoclays showed antimicrobial activity against
bacteria. Among them, the sample with HDTMA as the organic source and a
CEC load of 150 was the most efficient. The bacteria most susceptible
to these materials were \textit{E. coli}, \textit{S.
typhimurium}, and \textit{E. faecium}, which were the most
resistant. When the organoclays become magnetic, their antimicrobial
activity remains, and the samples have an advantage: they can be
recovered from the medium more easily than the non-magnetic ones, using
an external magnetic field. 

As a concluding remark, magnetic organoclays developed with a
relatively high amount of HDTMA can be used as biocide materials for
water treatment without releasing surfactants into the effluent.
Therefore, it was possible to manipulate the mud derived from the
treatment using an external magnetic field to separate the composites.

\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

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