Women in Science: Materials

Maria Chiara Bignozzi, Patricia Krawczak, Emilia Morallón, Ming Xu,
Jacqueline Anne Johnson

► To cite this version:

Maria Chiara Bignozzi, Patricia Krawczak, Emilia Morallón, Ming Xu, Jacqueline Anne Johnson. Women in Science: Materials. Maria Chiara Bignozzi; Patricia Krawczak; Emilia Morallón; Ming Xu; Jacqueline Anne Johnson. Frontiers Media SA, pp.445, 2021, 978-2-88966-951-6. 10.3389/978-2-88966-951-6 . hal-03281076

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WOMEN IN SCIENCE:
MATERIALS

EDITED BY: Maria Chiara Bignozzi, Patricia Krawczak, Emilia Morallon, Ming Xu and Jacqueline Anne Johnson

PUBLISHED IN: Frontiers in Materials and
Frontiers in Bioengineering and Biotechnology

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ISSN 1664-8714

ISBN 978-2-88966-951-6

DOI 10.3389/978-2-88966-951-6

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WOMEN IN SCIENCE:
MATERIALS

Topic Editors:

**Maria Chiara Bignozzi**, University of Bologna, Italy

**Patricia Krawczak**, IMT Lille Douai, France

**Emilia Morallon**, University of Alicante, Spain

**Ming Xu**, Huazhong University of Science and Technology, China

**Jacqueline Anne Johnson**, University of Tennessee Space Institute (UTSI),
United States

The *Frontiers in Materials* Editorial Office team are delighted to present the inaugural
"Women in Science: Materials" article collection, showcasing the high-quality work
of women in science across the breadth of materials science and engineering.

All researchers featured within this collection were individually nominated by the Topic Editors in recognition of their status as leading academics who have great potential to influence the future directions of their respective fields. The work presented here highlights the diversity of research performed across the entire breadth of the materials science and engineering field and presents advances in theory, experimentation, and methodology with applications for solving compelling problems.

This Editorial features the corresponding author(s) of each paper published within
this important collection, ordered by section alphabetically, highlighting them as
the great researchers of the future.

The *Frontiers in Materials* Editorial Office team would like to thank each researcher
who contributed their work to this collection. We would also like to personally thank
the Topic Editors for their exemplary leadership of this article collection; their strong
support and passion for this important, community-driven collection has ensured
its success and global impact.

Emily Young

Journal Development Manager
Topic Editors

Maria Chiara Bignozzi

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Prof. Maria Chiara Bignozzi (MCB) is an associate professor in Materials Science and Technology at the School of Engineering and Architecture, Alma Mater Studiorum University of Bologna, Italy, and director of Centro Ceramico (www.centroceramico.it). She is also involved in European and international standards, being a Convener of WG1 “Test methods” for ISO TC 189 and CEN TC 67 “Ceramic tiles”. Her research activity is mainly concerned with building materials (ceramic tiles, geopolymers/alkali-activated materials, concrete, cement, etc.) and their physical-mechanical performances, durability, and sustainability features.

Patricia Krawczak

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Prof. Patricia Krawczak is a Full Professor of Materials Science and Engineering at Ecole Nationale Supérieure Mines-Télécom Lille Douai, France. After being the director of the "Polymers and Composites Technology & Mechanical Engineering" department of this graduate school for 20 years, she has been the strategic program manager of "Technological Platforms" at the Institut Mines-Télécom since 2019. Her research expertise focuses on the advanced manufacturing, physics, and mechanics of plastics and composites. She has authored over 400 journal or conference publications and book chapters. She is a member of the Executive Board of the French society of plastics engineers and the Board of Directors of the French Industrial Technical Centre for polymer processing and composites. She is the recipient of the Légion d'Honneur national award.

Emilia Morallon

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Prof. Emilia Morallon is a Full Professor of Physical Chemistry at the University of Alicante, Spain, has been the Head of Electrocatalysis and Polymer Electrochemistry Research Group since 2008, and is a member of the Materials Institute of the University of Alicante. She has authored 240 papers and book
chapters and has supervised more than 20 Ph.D. theses. She has several research interests: preparation and characterization of conducting polymers, preparation and characterization of hybrid compounds, synthesis and characterization of metal oxides for use in environmental and energy applications, electrochemical characterization and electrochemical functionalization of carbon materials, and environmental and energy applications, such as fuel cells, supercapacitors, and the removal of pollutants.

## Ming Xu

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Prof. Ming Xu is a Full Professor of Huazhong University of Science and Technology (HUST), China, and the head of the lab of Carbon Nanomaterials of HUST (www.nanocarbon-hust.cn). Her research interests include synthesis and functionalization of carbon nanomaterials for dry adhesion, sensoring, energy storage and harvesting, processing of composite materials based on carbon nanomaterials, and a focus on the development of carbon nanomaterials application in extreme environments. She won the 7th Iijima Prize, which is recognized as one of the most distinguished honors in the field of carbon nanomaterial research.

## Jacqueline Anne Johnson

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Dr. Johnson completed her doctorate in solid-state physics in the research area of magnetic phase transitions at the University of Liverpool in 1985. She transitioned to working on glass materials after being approached by Pilkington Glass to solve technical problems. She was a Professor in Liverpool until 1995 when she joined Argonne National Laboratory in the United States. Here, she was introduced to solving the structure of amorphous materials using neutron scattering. After a 2-year period in administration, she returned to research to develop a new mammography system using a glass-ceramic plate. In 2007, Dr. Johnson returned to academia at the University of Tennessee Space Institute, and she continues to synthesize and characterize glasses, glass ceramics, and nanomaterials pertaining to medical devices, non-destructive evaluation, and image enhancement.

**Citation:** Bignozzi, M. C., Krawczak, P., Morallon, E., Xu, M., Johnson, J. A., eds. (2021). Women in Science: Materials. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88966-951-6
# Table of Contents

**09 Editorial: Women in Science: Materials**

Maria Chiara Bignozzi, Jacqueline Anne Johnson, Patricia Krawczak, Emilia Morallon and Ming Xu

## BIOMATERIALS

**13 Surface Triggered Self-Assembly of Fmoc-Tripeptide as an Antibacterial Coating**

Miryam Criado-Gonzalez, Muhammad Haseeb Iqbal, Alain Carvalho, Marc Schmutz, Loïc Jierry, Pierre Schaaf and Fouzia Boulmedais

**22 Evaluating the Toxicity of Ionic Liquids on Shewanella sp. for Designing Sustainable Bioprocesses**

Hakima Kebaili, Antonia Pérez de los Ríos, María José Salar-García, Víctor Manuel Ortiz-Martínez, Mostefa Kameche, Jesús Hernández-Fernández and Francisco J. Hernández-Fernández

## CARBON-BASED MATERIALS

**43 Thermal Properties Enhancement of Vertically Aligned Carbon Nanotubes-Based Metal Nanocomposites as Thermal Interface Materials**

Qiuhong Zhang, Brian A. Calderon, Charles R. Ebbing, Levi J. Elston, Larry W. Byrd and Bang-Hung Tsao

**55 Direct Graphene Synthesis on Lithium Niobate Substrate by Carbon Ion Implantation**

Yuhang Xu, Fei Lu, Kaijing Liu and Changdong Ma

**64 Effect of Co-solution of Carbon Precursor and Activating Agent on the Textural Properties of Highly Porous Activated Carbon Obtained by Chemical Activation of Lignin With $H_3PO_4$**

Imane Moulefera, Francisco J. García-Mateos, Abdelghani Benyoucef, Juana M. Rosas, José Rodríguez-Mirasol and Tomás Cordero

**78 Mechanically Robust Free-Standing Single-Walled Carbon Nanotube Thin Films With Uniform Mesh-Structure by Blade Coating**

Yuichi Kato, Atsuko Sekiguchi, Kazufumi Kobashi, Rajyashree Sundaram, Takeo Yamada and Kenji Hata

## CERAMICS AND GLASS

**87 Multifunctional Lightweight Mortars for Indoor Applications to Improve Comfort and Health of Occupants: Thermal Properties and Photocatalytic Efficiency**

Chiara Giosuè, Mattia Pierpaoli, Alessandra Mobili, Maria Letizia Ruello and Francesca Tittarelli

## COLLOIDAL MATERIALS AND INTERFACES

**99 Advances in Microtechnology for Improved Cytotoxicity Assessment**

Maite Garcia-Hernando, Fernando Benito-Lopez and Lourdes Basabe-Desmonts
**116** *Dynamic Wetting of Molten Polymers on Cellulosic Substrates: Model Prediction for Total and Partial Wetting*
Monica Francesca Pucci, Benoît Duchemin, Moussa Gomina and Joël Bréard

ENERGY MATERIALS

**129** *Energy Storage in Supercapacitors: Focus on Tannin-Derived Carbon Electrodes*
Jimena Castro-Gutiérrez, Alain Celzard and Vanessa Fierro

**154** *Non-precious Melamine/Chitosan Composites for the Oxygen Reduction Reaction: Effect of the Transition Metal*
B. Aghabarari, M. V. Martínez-Huerta, M. C. Capel-Sánchez and M. J. Lázaro

ENVIRONMENTAL MATERIALS

**164** *Experimental Study on an Innovative Biopolymeric Treatment Against Salt Deterioration of Materials in Cultural Heritage*
Mattia Bassi, Enrico Sassoni and Elisa Franzoni

MECHANICS OF MATERIALS

**188** *A Contribution to the Study of the Forming of Dry Unidirectional HiTape® Reinforcements for Primary Aircraft Structures*
Laure Bouquerel, Nicolas Moulin and Sylvain Drapier

**205** *An Incremental Cohesive Law for Delamination Under a Mixed Mode Loading*
Man Zhu, Larissa Gorbatikh and Stepan V. Lomov

**217** *Characterization of Surface Fatigue Crack Nucleation and Microstructurally Small Crack Growth in High Strength Aluminum Alloys*
Robert Fleishel, Cole Cauthen, Steven Daniewicz, Andrew Baker, J. Brian Jordon and Stephanie TerMaath

**233** *Changing Compliance of Poly(Vinyl Alcohol) Tubular Scaffold for Vascular Graft Applications Through Modifying Interlayer Adhesion and Crosslinking Density*
YeJin Jeong, Yuan Yao, Tizazu H. Mekonnen and Evelyn K. F. Yim

POLYMERIC AND COMPOSITE MATERIALS

**253** *Use of Flow Modeling to Optimize the Twin-Screw Extrusion Process for the Preparation of Lignocellulosic Fiber-Based Composites*
Françoise Berzin, Chantal David and Bruno Vergnes

**260** *Roles of Size, Shape, Amount, and Functionalization of Nanoparticles of Titania in Controlling the Tribo-Performance of UHMWPE Composites*
Meghashree Padhan, Gourab Paul and Jayashree Bijwe

**275** *Hollow Section Fibers Characterization for Seats Covers Fabric Application*
Valentina Brunella, Giulia Albini, Vito Guido Lambertini and Bartolomeo Placenza
**285** Under Glass Weathering of Hemp Fibers Reinforced Polypropylene Biocomposites: Degradation Mechanisms Based on Emitted Volatile Organic Compounds

Célia Badji, Jean-Marc Sotiropoulos, Joana Beigbeder, Hélène Garay, Anne Bergeret, Jean-Charles Bénézet and Valérie Desauziers

**297** Corrigendum: Under Glass Weathering of Hemp Fibers Reinforced Polypropylene Biocomposites: Degradation Mechanisms Based on Emitted Volatile Organic Compounds

Célia Badji, Jean-Marc Sotiropoulos, Joana Beigbeder, Hélène Garay, Anne Bergeret, Jean-Charles Bénézet and Valérie Desauziers

**298** Fiber Orientation Distribution Predictions for an Injection Molded Venturi-Shaped Part Validated Against Experimental Micro-Computed Tomography Characterization

María C. Quintana, Patricia M. Frontini, Aitor Arriaga, Bernhard Plank and Zoltan Major

**311** Processing-Structure-Properties of Cork Polymer Composites

Carla I. Martins and Vitória Gil

**323** Statistical Fatigue Investigation and Failure Prediction of a Healable Composite System

Nathan Hostettler, Amaël Cohades and Véronique Michaud

**332** Supramolecular Structure and Mechanical Properties of Wet-Spun Polyacrylonitrile/Carbon Nanotube Composite Fibers Influenced by Stretching Forces

Hamideh Mirbaha, Paolo Scardi, Mirco D'Incau, Shahram Arbab, Parviz Nourpanah and Nicola M. Pugno

**345** Role of Compatibilizer in Improving the Properties of PLA/PA12 Blends

Amulya Raj, Cedric Samuel and Kalappa Prashantha

**357** In-situ Monitoring of the Out-Of-Autoclave Consolidation of Carbon/ Poly-Ether-Ketone-Ketone Prepreg Laminate

Florence Saffar, Camille Sonnenfeld, Pierre Beauchêne and Chung Hae Park

## SMART MATERIALS

**370** Hydrogen Sulfide Detection by Sensors Based on Conductive Polymers: A Review

Caroline Duc, Mohamed-Lamine Boukhenane, Jean-Luc Wojkiewicz and Nathalie Redon

## STRUCTURAL MATERIALS

**383** Gender Balance in Construction Material Research: The Analysis of Alkali-Activated Materials by a Bibliometric Study Using Scopus Database

Giulia Masi, Stefania Manzi and Maria Chiara Bignozzi

**394** A Case Study of Industrial Symbiosis to Reduce GHG Emissions: Performance Analysis and LCA of Asphalt Concretes Made With RAP Aggregates and Steel Slags

Alessandra Bonoli, Anna Degli Esposti and Chiara Magrini

**408** 3D-Extrusion Manufacturing of a Kaolinite Dough Taken in Its Pristine State

Séverine A. E. Boyer, Lucie Jandet and Alain Burr
420 **Characterization of Fly Ash Alkali Activated Foams Obtained Using Sodium Perborate Monohydrate as a Foaming Agent at Room and Elevated Temperatures**
Lidija Korat and Vilma Ducman

435 **Alkali-Activated Binders From Waste Incinerator Bottom Ashes and Metakaolin Reinforced by Recycled Carbon Fiber Composites**
Stefania Manzi, Isabella Lancellotti, Giulia Masi and Andrea Saccani
# Editorial: Women in Science: Materials

Maria Chiara Bignozzi<fnref n="1" />*, Jacqueline Anne Johnson²*, Patricia Krawczak³*, Emilia Morallon⁴* and Ming Xu⁵*

**Keywords:** women in STEM, women in science and engineering, materials engineering, materials science, gender equality

## Editorial on the Research Topic

### Women in Science: Materials

According to UNESCO Institute for Statistics (UIS) data (2019), <30% of researchers worldwide are women. Long-standing biases and gender stereotypes are discouraging girls and women away from science-related fields. In particular, STEM (science, technology, engineering, and mathematics) research is traditionally male dominated and women remain underrepresented. As UNESCO has highlighted, both science and gender equality are, however, essential to ensuring sustainable development. To change traditional mindsets, gender equality must be promoted, stereotypes defeated, and girls and women should be encouraged to pursue careers in STEM.

Throughout history, the contributions of female researchers to scientific progress have been extremely important. Yet, whereas the extraordinarily talented Nobel laureate Marie Skłodowska-Curie is often mentioned as a role model, it is nowadays well-established that countless women such as Lise Meitner have received less recognition and acknowledgment for their research findings than their male counterparts [so-called Matilda effect (Rossiter, 1993)].

Also, women traditionally publish less than men according to the European Commission She Figures, the ratio of women to men among authors of scientific publications in the EU being on the average one to two (European Union, 2019), and recent surveys are highlighting a further drop in academic submissions from female scientists since the start of the Covid-19 pandemic early 2020.

Role models are definitely extremely important to show to younger generations the growing impact of female researchers to science. Therefore, continuing the spirit of the International Day of Women and Girls in Science, *Frontiers in Materials* is proud to offer this platform to promote the work of female scientists across the breadth of materials science and engineering.

This inaugural "Women in Science: Materials" collection (Figure 1) aims to highlight the impact of women researchers working in materials science and engineering. It gathers a selection of original articles with the lead author and/or corresponding author being a woman.

A total of 32 contributions (three reviews, one brief research report, and 28 original research articles) present advances in theory, experiment, and methodology with applications to compelling problems, across almost all sections of the journal.

## OPEN ACCESS

**Edited and reviewed by:**

Alberto Corigliano,
Politecnico di Milano, Italy

*Correspondence:

Maria Chiara Bignozzi
maria.bignozzi@unibo.it
Jacqueline Anne Johnson
jjohnson@utsi.edu
Patricia Krawczak
patricia.krawczak@imt-lille-douai.fr
Emilia Morallon
morallon@ua.es
Ming Xu
ming.xu@hust.edu.cn

**Specialty section:**

This article was submitted to
Mechanics of Materials,
a section of the journal
Frontiers in Materials

**Received:** 15 February 2021

**Accepted:** 25 February 2021

**Published:** 23 April 2021

**Citation:**

Bignozzi MC, Johnson JA,
Krawczak P, Morallon E and Xu M
(2021) *Editorial: Women in Science:
Materials*. Front. Mater. 8:668092.
doi: 10.3389/fmats.2021.668092

<footnote n="1">¹ Department of Civil, Chemical, Environmental and Materials Engineering, University of Bologna, Bologna, Italy, ² University of Tennessee, Space Institute, Tullahoma, TN, United States, ³ IMT Lille Douai, Institut Mines-Télécom, Univ. Lille, Centre for Materials and Processes, Douai, France, ⁴ Physical Chemistry Department, Institute of Materials Science, University of Alicante, Alicante, Spain, ⁵ School of Materials Science and Engineering, Huazhong University of Science and Technology, Wuhan, China</footnote>
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frontiers
in Materials

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Women in
Science:
Materials

<caption>**FIGURE 1** | "Women in Science: Materials" collection in *Frontiers in Materials*.</caption>

* Mechanics of materials (Bouquerel et al.; Jeong et al., TerMaath et al.; Zhu et al.)

* Smart materials (Duc et al.)

* Polymeric and composite materials (Badji et al.; Berzin et al.; Brunella et al.; Hostettler et al.; Martins and Gil; Mirbaha et al.; Padhan et al.; Quintana et al., Raj et al.; Saffar et al.)

## REFERENCES

European Union (2019). *European Commission She Figures 2018*. Publications Office of the European Union, Luxembourg. doi: 10.2777/936

Rossiter, M.W. (1993). The Matthew Matilda effect in science. *Soc. Stud. Sci.* 23, 325–341. doi: 10.1177/03063129302302004

UNESCO Institute for Statistics (2019). *Women in Science (n° 55)* [Fact sheet], UIS/FS/2019/SCI/55, UIS Publ., Montreal, QC, Canada. Available online at: https://unesdoc.unesco.org/ark:/48223/pf0000370742

* Carbon-based materials (Kato et al.; Moulefera et al.; Xu et al., Zhang et al.)

* Structural materials (Bonoli et al.; Boyer et al.; Korat and Ducman; Manzi et al., Masi et al.)

* Colloidal materials and interfaces (Garcia-Hernando et al.; Pucci et al.)

* Energy materials (Castro-Gutiérrez et al.)

* Ceramics and glass (Giosuè et al.)

* Environmental materials (Bassi et al.)

* Biomaterials (Criado-Gonzalez et al.; Kebaili et al.)

The Guest Editorial team hope that this collection of papers will be the foundation of an international network of women researchers working in materials science and engineering, and a starting point for future collaborations and discussions.

## AUTHOR CONTRIBUTIONS

This Editorial was jointly written by all co-authors who also served as Guest Editors for the Research Topic. All authors contributed to the article and approved the submitted version.

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Bignozzi, Johnson, Krawczak, Morallon and Xu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Biomaterials

<figure><img src="image_11.png" /><figcaption>Fouzia Boulmedais</figcaption></figure>

Dr. Fouzia Boulmedais is a physical chemist with a CNRS position at Institut Charles Sadron, Strasbourg; here, she develops biopolymer films as biomaterial coatings and electrodeposited polymeric films for biosensing. She graduated from University Louis Pasteur, Strasbourg, where she received her Ph.D. in chemistry and physical chemistry in 2003. In 2004, she worked as a post-doc with Prof. Marcus Textor at ETH Zurich, Switzerland, and with Prof. Gleb Sukhorukhov at Max Planck Institute, Germany, on electrodissolution of polyelectrolytes multilayers. As of 2006, she has been a co-author of 109 publications and four patents.

<figure><img src="image_12.png" /><figcaption>Miryam Criado-Gonzalez</figcaption></figure>

Dr. Miryam Criado-Gonzalez studied Chemical Engineering (2011) at the University of Salamanca, Spain, where she also pursued a Master of Chemical Engineering (2013). Then, she studied for a Master of Polymer Science (2014) at the Institute of Polymer Science and Technology (ICTP-CSIC) in Madrid, Spain, where she also obtained her Ph.D. (2017). She held a postdoctoral position at Institut Charles Sadron (CNRS) in collaboration with INSERM (Strasbourg, France) in 2017. From June 2020, she worked as a postdoctoral researcher on a collaborative project CSIC-CNRS at ICTP, Madrid, Spain. In March 2021, she joined the Innovative Polymers Group at POLYMAT, Spain. Her research interests include the layer-by-layer assembly of biopolymers, supramolecular peptide/polymer hydrogels, and additive manufacturing of conducting polymers.

<figure><img src="image_13.png" /><figcaption>María José Salar</figcaption></figure>
Dr. María José Salar is an assistant professor at the Technical University of Cartagena, Spain. She got her BSc (2010) and MSc (2011) in Chemical Engineering and Molecular Chemistry, respectively, at the University of Murcia, Spain. Following a move to the Technical University of Cartagena in 2012, she obtained her Ph.D. and the extraordinary Ph.D. award in 2016. After her Ph.D., she was funded by the Seneca Foundation to conduct her research at the Bristol BioEnergy Centre, United Kingdom. Her research field focuses on the design and optimization of microbial fuel cells as well as the use of green solvents, such as ionic liquids, in membrane technology. These research lines have resulted in more than 45 scientific publications (h-index: 17) and nine book chapters.
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Check for
updates

# Surface Triggered Self-Assembly of Fmoc-Tripeptide as an Antibacterial Coating

Miryam Criado-Gonzalez¹,²*, Muhammad Haseeb Iqbal¹,², Alain Carvalho¹,
Marc Schmutz¹, Loïc Jierry¹, Pierre Schaaf¹,²,³ and Fouzia Boulmedais¹*

¹ Université de Strasbourg, CNRS, Institut Charles Sadron UPR 22, Strasbourg, France, ² Institut National de la Santé et de la Recherche Médicale, UMR-S 1121, "Biomatériaux et Bioingénierie", Strasbourg, France, ³ Faculté de Chirurgie Dentaire, Fédération de Médecine Translationnelle de Strasbourg and Fédération des Matériaux et Nanoscience d'Alsace, Université de Strasbourg, Strasbourg, France

In western countries, one patient on twenty will develop a nosocomial infection during his hospitalization at health care facilities. Classical antibiotics being less and less effective, this phenomenon is expanding year after year. Prevention of bacteria colonization of implantable medical devices constitutes a major medical and financial issue. In this study, we developed an antibacterial coating based on self-assembled Fmoc-tripeptide. Fmoc-FFpY peptides (F: phenylalanine; Y: tyrosine; p: PO₄²⁻) are dephosphorylated enzymatically into Fmoc-FFY by action of alkaline phosphatase functionalized silica nanoparticles (NPs@AP), previously deposited on a surface. Fmoc-FFY peptides then self-assemble through π-π stacking interactions, hydrogen bonds and hydrophobic interactions adopting β-sheets secondary structures. The obtained hydrogel coatings show fibrillary structures observed by cryo-scanning electron microscopy with a thickness of few micrometers. At low concentration (≤0.5 mg.mL⁻¹), self-assembled Fmoc-FFY has a superior antibacterial activity than Fmoc-FFpY peptide in solution. After 24 h of incubation, Fmoc-FFY hydrogel coatings fully inhibit the development of Gram-positive *Staphylococcus aureus* (*S. aureus*). The antibacterial effect is maintained on an *in vitro* model of repetitive infection in the case of *S. aureus*. This coating could serve in infections were Gram positive bacteria are prevalent, e.g., intravascular catheter infections. This work gives new insights toward the design of an alternative antimicrobial coating.

**Keywords:** peptides, hydrogels, antimicrobial, enzyme-assisted self-assembly, nanoparticles

## INTRODUCTION

Biomedical implants, i.e., prosthetics, catheters or intraocular lenses, are indispensable in medicine and gain increasing attention over the years (Lombardi et al., 2019). However, the direct contact of their surfaces with biological fluids becomes susceptible to bacterial colonization and biofilm formation leading to a major medical and financial issue (McCloskey et al., 2014). Implant-associated infections increase the probability of implant failure and may result, if untreated, in chronic microbial infection, inflammation, tissue necrosis, and even morbidity (McCloskey et al., 2014). In western countries one patient on twenty will develop a nosocomial infection during

**OPEN ACCESS**

**Edited by:**

Patricia Krawczak,
IMT Lille Douai, France

**Reviewed by:**

Garry Laverty,
Queen's University Belfast,
United Kingdom
Bing Xu,
Brandeis University, United States

**\*Correspondence:**

Miryam Criado-Gonzalez
miryamcg22@gmail.com
Fouzia Boulmedais
fouzia.boulmedais@ics-cnrs.unistra.fr

**Specialty section:**

This article was submitted to
Biomaterials,
a section of the journal
Frontiers in Bioengineering and
Biotechnology

**Received:** 13 May 2020

**Accepted:** 20 July 2020

**Published:** 07 August 2020

**Citation:**

Criado-Gonzalez M, Iqbal MH, Carvalho A, Schmutz M, Jierry L, Schaaf P and Boulmedais F (2020) Surface Triggered Self-Assembly of Fmoc-Tripeptide as an Antibacterial Coating. *Front. Bioeng. Biotechnol.* 8:938. doi: 10.3389/fbioe.2020.00938
his hospitalization at health care facilities (Cassini et al., 2016). These infections are mostly due to *Staphylococcus epidermidis*, in the case of intravascular catheter-associated infections (Rupp and Archer, 1994), and *Staphylococcus aureus* (S. aureus) in the case of metallic implants (Barth et al., 1989). Biofilm formation contributes to the resistance to antibiotic treatments, it protects the bacterial colonies from host defense systems and bactericidal agents (Kaplan, 2011). This leads to the chemotherapeutic failure which often results in an increase of the resistance mechanism adopted by many bacterial strains, particularly those involving S. aureus (Darouiche, 2004). In this scenario, the development of antimicrobial coatings to protect against such infections has become a major field of scientific and technological research (Séon et al., 2015). As alternative to antibiotics, antimicrobial peptides are promising candidates to overcome pathogen resistance and for clinical exploitation (Lombardi et al., 2019). Self-assembly of peptides, with a sequenced-defined chemical structure, can give hydrogels with a desired functionality which becomes increasingly attractive in the development of therapeutics materials (Makam and Gazit, 2018; Criado-Gonzalez et al., 2020a).

In the last decade, low molecular weight hydrogelators (LMWH) fabricated from natural biomolecules such as amino acids got great interest due to their ease and powerful bottom-up fabrication. They self-assemble into supramolecular hydrogels by non-covalent interactions, i.e., electrostatic, van der Waals forces, hydrogen bonding and π-π stacking (McCloskey et al., 2014), in response to external stimuli, such as temperature change (Collier et al., 2001), pH switch (Aggeli et al., 2003; Chen et al., 2011), solvent change (Mahler et al., 2006), electrostatic interactions (Criado-Gonzalez et al., 2020b), chemical (Bowerman and Nilsson, 2010; Zhao et al., 2011) or enzymatic reactions (Yang et al., 2004; Yang and Xu, 2006; Criado-Gonzalez et al., 2019b). Ultrashort peptides, up to 7 amino acids in length, are an emerging class of LMWH of increasing interest due to their versatility in molecular design, ease synthesis, and costs reduction. Few studies have been carried out on the development of self-assembling materials with antibacterial properties using short peptides. Based on the pioneering work of Moir and coll. (Soscia et al., 2010; Kumar et al., 2016) which demonstrated the antimicrobial activity of an amyloid-β peptide, Schnaider et al. (2017) proved the antibacterial activity of a self-assembled dipeptide, diphenylalanine (FF), against *Escherichia Coli* (*E. coli*). In this work, FF moiety was identified as the central recognition module of amyloid, as well as the fundamental self-assembly motif proving the efficiency of this unit for the development of self-assembling antimicrobial hydrogels. Fluorenylmethyloxycarbonyl-FF (Fmoc-FF) derived peptides were shown to possess excellent activity against the most antibiotic resistant biofilm phenotype of both Gram-positive and Gram-negative bacteria (McCloskey et al., 2017). Fmoc-FF self-assemblies were employed as host hydrogels to incorporate silver nanoparticles to improve the antimicrobial response (Paladini et al., 2013). The backbone of this dipeptide was modified incorporating an urea moiety to inhibit the *E. coli* bacterial adhesion (Basavalingappa et al., 2019).

<figure><img src="image_15.png" /></figure>

Poorly soluble in water, Fmoc-FF peptide self-assembly is induced by dissolution in DMSO followed by a dilution step in water (Basavalingappa et al., 2019), by change of pH (Paladini et al., 2013), or by heating the solution, up to 90°C for solubilization, followed by a cooling step (Kumar et al., 2016). To overcome these tedious processes, the enzyme assisted self-assembly (EASA) of peptides was introduced by Xu and co-workers (Yang et al., 2004; Yang and Xu, 2006). Soluble at room temperature in aqueous solution, phosphorylated peptides were transformed into gelators by alkaline phosphatase (AP) catalyzing the removal of the phosphate groups. Later, we introduced the use of non-self-assembling Fmoc-FFpY peptide (Y: tyrosine; p: $PO_4^{2-}$), which is transformed into the hydrogelator Fmoc-FFY by AP. The enzymatic assisted self-assembly of Fmoc-FFpY was localized on a planar substrate (Vigier-Carrière et al., 2017) or on silica nanoparticles (Criado-Gonzalez et al., 2019a) thanks to the functionalization of their surfaces by AP. In contrast to the pH triggered self-assembly (Jayawarna et al., 2006; Raeburn et al., 2012), the EASA allows to use a more water-soluble peptide as a precursor and to obtain, by simple contact with the peptide solution, a fast and localized self-assembly on the surface of a substrate, previously functionalized by the enzyme.

Integrating self-assembling peptides with antimicrobial property on the surface of a biomaterial is an innovative design for the development of antibacterial materials. Herein, we report an antibacterial coating based on Fmoc-FFY hydrogel self-assembled by enzymatic dephosphorylation. To this aim, AP functionalized silica nanoparticles (NPs@AP) were first deposited on the surface of a material using the layer-by-layer method and then put in contact with Fmoc-FFpY solution to obtain the Fmoc-FFY hydrogel coating (Scheme 1). NPs@AP allowed to immobilize a higher quantity of enzyme on the surface than AP monolayer. The antimicrobial property was tested against *S. aureus*, Gram-positive strain which is one of the most virulent bacteria leading to high rates of device-related systemic infections and mortality and *E. coli*, a Gram-negative strain mainly found on the surface of urinary catheters.
# MATERIALS AND METHODS

## Materials

Poly(ethylene imine) (PEI, Mw = 250 000 g.mol⁻¹), Poly(allylamine hydrochloride) (PAH, Mw = 58 000 g.mol⁻¹), Alkaline Phosphatase (AP, 10 DEA units.mg protein⁻¹) from bovine intestinal mucosa, Poly(sodium 4-styrenesulfonate) (PSS, Mw = 70 000 g.mol⁻¹), Tetraethyl orthosilicate (TEOS), (3-glycidyloxypropyl) trimethoxysilane (GPMS) were from Sigma Aldrich. p-Nitrophenyl phosphate (PNP) was from ThermoFisher Scientific. Sodium tetra-borate anhydrous (borax) and dry toluene were supplied by Acros Organics. Fmoc-FFpY was purchased by PepMic, ammonium hydroxide by Carlo Erba and ethanol by VWR. Mueller Hinton (MH) broth was purchased from Merck (Germany).

## Synthesis and Functionalization of Silica Nanoparticles

The synthesis of silica nanoparticles (NP) and their functionalization with AP were carried out following the procedure described previously (Criado-Gonzalez et al., 2019a). After their synthesis, NPs were functionalized covalently by AP using an epoxy silane coupling agent, giving rise to NPs@AP (Supplementary Data). The catalytic activity of NPs@AP suspensions was determined employing PNP, a substrate which is transformed in para-nitrophenol (λmax = 405 nm) by action of AP.

## Multilayer Film Preparation and Fmoc-FFpY Self-Assembly

All solutions were prepared in 25 mM borax buffer pH 9.5. Multilayer films were built on glass slides of 12 mm diameter (Marienfeld) or silicon slides of 10 × 10 mm (Sil'tronix Silicon Technologies). As the substrates are negatively charged, a precursor layer of branched PEI (1 mg.mL⁻¹) was deposited to achieve a homogeneous distribution of positive charges over the surface allowing an efficient deposition of the coating. For that, substrates were immersed in a solution of PEI for 5 min followed by a rinsing step with borax buffer for 2 min. After that, the multilayer film was built up through sequential contact of the substrate with 400 µL of PSS (1 mg.mL⁻¹) (polyanion) or PAH (1 mg.mL⁻¹) (polycation) solutions for 5 min with a rinsing step in borax buffer of 2 min after each polyelectrolyte deposition. This cycle was repeated two times and samples were denoted as polyelectrolyte multilayer (PEM). Then the substrate was put in contact with 400 µL of NPs@AP (1.25% w/v in borax buffer) suspension for 60 min followed by three rinsing steps of 2 min in borax buffer. Samples were denoted as NPs@AP coatings. Subsequently, 400 µL Fmoc-FFpY (1 mg.mL⁻¹) solution was brought in contact with NPs@AP coating for 16 h.

## Quartz Crystal Microbalance With Dissipation Monitoring (QCM-D)

Quartz crystal microbalance with dissipation monitoring experiments were performed in a QCM-D cell on a Q-Sense E1 apparatus (Q-Sense AB, Gothenburg, Sweden) at 22°C

using an open cell. The resonance frequencies of a gold coated crystal and the dissipation factors at the fundamental frequency at 5 MHz (ν = 1) were monitored during the deposition of NPs@AP and the Fmoc-FFY self-assembly. The perturbation due to the pipetting of the solutions at each deposition steps were removed from the plot.

## Atomic Force Microscopy (AFM)

Atomic force microscopy Multimode Nanoscope IV (Bruker, Palaiseau, France) was used to analyze the surface topography of the coatings. Micrographs were recorded in Peak Force Tapping (ScanAsyst) mode by using silicon tips from Bruker (Model: ScanAsyst-Air) mounted on aluminum coated silicon nitride cantilevers. All samples were observed in dry state with triangular cantilevers having a spring constant of around 0.4 N.m⁻¹ and a nominal tip radius of 2 nm. Selected AFM images were treated with the NanoScope Analysis Software (version 1.7). Samples were prepared on silicon substrates and air dried before analysis. Prior to the sample preparation, the silicon wafers were incubated in ethanol/water (50% by vol) mixture for 15 min and activated using plasma treatment for 3 min.

## Circular Dichroism (CD)

Circular dichroism (CD) spectra were recorded between 190 and 320 nm using a Jasco J-1100 spectropolarimeter with a data pitch of 1 nm on the light wavelength. Samples were built up on a quartz slide of 1 mm thickness.

## Fluorescence Spectroscopy

Fluorescence spectra were recorded between 300 and 355 nm using a Fluoromax-4 (Horiba Jobin Yvon - Edison, NJ, United States) at an excitation wavelength of 290 nm using a quartz slide of 1 mm thickness to build up the samples.

## Scanning Electron Microscopy (SEM) and Cryo-SEM

The Fmoc-FFY hydrogel coating on a Silicon wafer was placed on a home-made cryo-holder (Vigier-Carrière et al., 2017) to be quickly plunged into an ethane slush. As the sample is free standing over the holder, the sample is rapidly frozen during the plunging by direct contact with the liquid ethane. Subsequently, the sample is transferred into the Quorum PT 3010 chamber attached to the microscope. There, the frozen sample is fractured with a razor blade. A slight etching at -90°C may be performed to render the fibers more visible. The sample is eventually transferred in the FEG-cryo SEM (Hitachi SU8010) and observed at 1 kV at -150°C.

## Inverted Tube Tests

All solutions were prepared in 25 mM borax buffer pH = 9.5. 150 µL of Fmoc-FFpY (1 mg.mL⁻¹) was mixed with 50 µL of NPs@AP (5% w/v) in vials. After 24 h, inverted tube tests were carried out to determine the hydrogel formation. Then, 400 µL of bacteria culture medium (MH) was added and the stability was checked up to 9 days.
## Antibacterial Activity

Antibacterial tests were carried out employing one strain Gram-positive bacteria, *Staphylococcus aureus* (S. aureus, ATCC 25923) and another strain Gram-negative bacteria, *Escherichia coli* (E. coli, ATCC 25922). S. aureus and E. coli were precultured separately in aerobic conditions at 37°C in a MH broth medium (Merck, Germany), pH 7.4. One colony from previously prepared agar plates by bacteria streaking protocol was transferred to 7 mL of MH medium and incubated in an agitator overnight at 37°C. To obtain bacteria in their mid-logarithmic phase of growth, the absorbance at 620 nm (OD$_{620}$) of overnight cultures was adjusted to 0.001 by diluting in MH, corresponding to a final cell density of approximately $8 \times 10^5$ CFU.mL$^{-1}$. Cultures growing in the presence of antibiotics (Tetracycline and Cefotaxime) were taken as positive control. Bacteria quantification (in colony forming unit per mL, CFU.mL$^{-1}$) was performed at time zero and 24 h after the incubation with the samples. This was determined by plating 100 µL of the supernatant, after serial dilution, on nutrient agar plates at 37°C overnight, then the viable cell colonies were counted and represented as $log_{10}$ reduction (CFU.mL$^{-1}$). To determine the Minimum Inhibitory Concentration (MIC) value of Fmoc-FFpY and Fmoc-FFY hydrogel, the peptide was dissolved in RPMI medium at 2 mg.mL$^{-1}$ and at lower concentrations by serial dilution in RPMI. S. aureus was pre-cultured overnight in MH medium, and diluted in RPMI medium. MIC value of Fmoc-FFpY was determined by mixing 100 µL of the peptide solution and 100 µL of S. aureus suspension with a final optical density of OD = 0.001. To form the Fmoc-FFY hydrogel, 100 µL of the peptide solution (at different concentrations) was mixed with 100 µL of AP solution (1.4 µg.mL$^{-1}$) and left 24 h at 22°C. MIC value of Fmoc-FFY hydrogel was determined by adding to the hydrogel 200 µL of S. aureus inoculation with a final optical density of OD$_{620}$ = 0.001. Fmoc-FFY hydrogel coating samples were built up on glass substrates of 12 mm diameter, placed in 24-well plates and sterilized for 20 min by UV light. 400 µL of S. aureus or E. coli inoculation (OD$_{620}$ = 0.001) were added to each well and incubated at 37°C for 24 h. In repetitive culture experiments, the supernatant was totally removed and replaced by 400 µL of freshly prepared S. aureus or E. coli inoculation (OD$_{620}$ = 0.001). Regarding the statistical analysis, all experiments were carried out independently in triplicate and three analyses per replication at least were done. The significant differences in the experimental data were analyzed using the ANOVA procedure (SAS Institute Inc., Cary, NC, United States) at *p* < 0.01 with mean separation determined by Tukey's multiple range tests.

## Cell Cytotoxicity Test

Cytotoxicity assays were carried out by incubating the gels with 1 mL of DMEM at 37°C. After 24 and 48 h, the extracts were removed under sterile conditions. Separately, NIH 3T3 mouse embryonic fibroblasts cells were seeded at a density of $1 \times 10^5$ cells.mL$^{-1}$ in complete medium in a sterile 24 well culture plate and incubated to confluence. After 24 and 48 h of incubation, the medium was replaced with the corresponding extracts and incubated at 37°C in humidified air with 5% CO$_{2}$ for 24 h and

48 h. Subsequently, plates were incubated with 500 µL per well of a MTT solution (0.1% w/v 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide in Phosphate Buffer Saline) and incubated for 180 min at 37°C. Medium was displaced by 500 µL of DMSO. Optical Density (OD) was measured at 570 nm. The cell viability was calculated from Eq. (1):

$$ \text{Cell viability (\%)} = \frac{OD_S - OD_B}{OD_C - OD_B} \times 100 \quad (1) $$

where OD$_{S}$, OD$_{B}$, and OD$_{C}$ are the optical density for the sample (S), blank (culture medium) (B), and control (glass) (C), respectively.

## RESULTS AND DISCUSSION

### Physico-Chemical Characterization of Fmoc-FFY Hydrogel Coating

Alkaline phosphatase functionalized silica nanoparticles suspensions were obtained with a solid content of 5% (w/v) and had an average diameter of $132 \pm 10$ nm (Supplementary Figure S1). The suspension showed an activity equivalent to 30 units.mL$^{-1}$ determined using PNP, a model substrate of AP, with a calibration curve (Supplementary Figure S2). To immobilize NPs@AP on a planar surface, a PEI-(PSS/PAH)$_2$ polyelectrolyte multilayer film (PEM) was first deposited as precursor film. When NPs@AP suspension is put in contact with the PEM precursor film, a high increase of the normalized frequency shift is observed by QCM-D reaching about 700 Hz after the rinsing steps (Figure 1A).

The obtained NPs@AP coating has a catalytic activity equivalent to $0.6 \mu g.mL^{-1}$ of AP in solution, corresponding to $0.08 \mu g.cm^{-2}$ of active enzyme. AFM images of NPs@AP coating showed round shape NPs all over the surface (Supplementary Figure S3). The section profile of the AFM images provides an average diameter of the deposited NPs of 140 nm (Supplementary Figure S3C) in agreement with SEM observations (Supplementary Figure S1). The NPs@AP coating was then employed as catalytic support for the localized growth of supramolecular peptide by putting it in contact with Fmoc-FFpY solution ($1 \text{ mg.mL}^{-1}$) (Scheme 1). The Fmoc-FFY self-assembly induced an increase of the frequency shift of about 230 Hz after 3.5 h (Figure 1A). The dissipation value reached $400 \times 10^{-6}$ which is the signature of highly hydrated films (Figure 1B). In contrast, NPs@AP coating had a dissipation value of $130 \times 10^{-6}$. By decreasing Fmoc-FFpY concentration of the solution in contact with NPs@AP coating, the final frequency shift decreased reaching 100 Hz at 0.5 and $0.25 \text{ mg.mL}^{-1}$ and 50 Hz at 0.1 mg.mL$^{-1}$. This was corroborated by the decrease of the dissipation value (Supplementary Figures S4A,B). As a control experiment, the contact of a non-self-assembling peptide, Fmoc-G-OH ($1 \text{ mg.mL}^{-1}$), with the NPs@AP coating induces an increase of 43 Hz of the frequency shift (Supplementary Figures S4C,D). Fmoc-G-OH peptide is probably physisorbed on the surface such as Fmoc-FFpY when the concentration is lower than 0.1 mg.mL$^{-1}$.
<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /><figcaption>**FIGURE 1** | Evolution of the (A) fundamental frequency shift and (B) dissipation value, measured by QCM-D at 5 MHz, during the deposition of NPs@AP on PEM precursor film, leading to NPs@AP coating, followed by the contact with Fmoc-FFpY solution leading to the Fmoc-FFY hydrogel coating. (C) Circular Dichroism and (D) Fluorescence emission (λ<sub>ex</sub> = 290 nm) spectra of Fmoc-FFY hydrogel coating (black curve) and Fmoc-FFpY solution (blue curve).</figcaption></figure>

The structural organization of Fmoc-FFY self-assembled on NPs@AP coating, named Fmoc-FFY hydrogel coating, was investigated by CD (Figure 1C and Supplementary Figure S5). The CD spectrum shows a positive band at 196 nm and a strong negative band at 215 nm characteristics of β-sheets structures (Smith et al., 2008). The presence of a negative band at 270 nm is assigned to offset face-to-face stacking of the Fmoc moieties which is not observed in the case of non-self-assembling Fmoc-FFpY in solution (Ryan et al., 2010). The excimer formation of Fmoc moieties was checked after Fmoc-FFY self-assembly by fluorescence spectroscopy (Tang et al., 2011; Criado-Gonzalez et al., 2019b). When excited at 290 nm, Fmoc-FFpY solution have a fluorescence emission peak at 312 nm due to fluorenyl moieties (Figure 1D). The self-assembly of Fmoc-FFY on NPs@AP coating induced a shift of this peak up to 326 nm, due to fluorenyl excimers.

Morphological Characterization of
Fmoc-FFY Deposited Hydrogel

The morphology of the Fmoc-FFY hydrogel coating was
visualized by Cryo-SEM (Figure 2). The cross-section of the
coating showed three different areas. The magnification of the

bottom part allowed to distinguish monolayer and multilayers
of round shape NPs@AP, with a size of ~ 120 nm determined
using ImageJ, on the surface of the glass slide (Figure 2B). It can
be noticed that there is no diffusion of NPs@AP throughout the
whole Fmoc-FFY hydrogel after 16 h in contact (dashed line in
Figure 2B). Fmoc-FFY fibers grow from the NPs@AP giving rise
to a hydrogel coating with a thickness of ~ 5 µm. A gradient
of organic matter is observed in the self-assembled Fmoc-FFY
hydrogel with two different areas (Figure 2A). Higher density
of fibers is close to the bottom, in direct contact NPs@AP, and
a lower density on the upper part of the Fmoc-FFY hydrogel.
A magnification of the upper part allows to distinguish long
fibers of micrometer length oriented in the vertical direction
perpendicular to the substrate (Figure 2C). Fmoc-FFY fibers have
a diameter ranging from 13 to 40 nm (red arrows in Figure 2C).

Antimicrobial Properties of Fmoc-FFY
Hydrogel Coating

Before to study the antibacterial properties of the Fmoc-
FFY hydrogel coating, the stability of the hydrogel was tested
in contact with bacteria culture medium. For that purpose,
<figure><img src="image_20.png" /><figcaption>**FIGURE 2** | Cryo-SEM images of (A) Fmoc-FFY hydrogel self-assembled on NPs@AP coating, (B) zoom-in of the bottom area to show the localization of the NPs@AP delimited with a dashed line and highlighted with red arrows and (C) zoom-in of the upper part showing Fmoc-FFY fibers (red arrows).</figcaption></figure>

Fmoc-FFY hydrogel was formed in a vial by mixing Fmoc-FFpY solution and NPs@AP suspension. The inverted tube test indicated the formation of a gel after 24 h (Supplementary Figure S6). The hydrogel remains stable up to 9 days in contact with MH culture medium. To underline the necessity of the enzymatic dephosphorylation of the peptide, the antibacterial properties of Fmoc-FFpY and Fmoc-FFY hydrogel were studied against Gram-positive S. aureus in RPMI media at different concentrations in peptide (Figure 3A). Bacteria quantification (in colony forming unit per mL, CFU.mL⁻¹) was performed at time zero and 24 h after the incubation with the samples and presented in log₁₀ reduction. At 1 mg.mL⁻¹, 3-log₁₀ (99%) reduction bacterial load against S. aureus is obtained for Fmoc-FFpY peptide, whereas 4-log₁₀ (99.99%) reduction were achieved with Fmoc-FFY hydrogel, obtained by dephosphorylation of the peptide. At lower peptide concentrations (≤0.5 mg.mL⁻¹), the antibacterial property of Fmoc-FFpY is lost whereas Fmoc-FFY hydrogel showed 3-log₁₀ (99%) reduction of S. aureus suspension up to 0.1 mg.mL⁻¹ of peptide. The self-assembly of the peptide ensure the antibacterial effect at low concentration. Bare NPs@AP coatings did not prevent the proliferation of the bacteria (Supplementary Figure S7). The antibacterial effect of the Fmoc-FFY hydrogel coating, self-assembled on NPs@AP at different concentrations in Fmoc-FFpY, was tested against S. aureus (Figure 3B). After 24 h of contact, Fmoc-FFY hydrogel coatings demonstrated efficacy of more than 4-log₁₀ (99.99%) reduction of bacterial load against S. aureus whatever the concentration of peptide used for their self-assembly up to 0.1 mg.mL⁻¹. To mimic the worst conditions of repetitive bacterial infections, for example in the case of urinary or venous catheter-associated infections, the bacterial inoculation in contact with the hydrogel coating was completely renewed every 24 h. At least 3-log₁₀ (99%) reduction of S. aureus was observed for the second culture in contact with Fmoc-FFY hydrogel coating self-assembled with 1 mg.mL⁻¹ Fmoc-FFpY. At lower concentration in peptide, the second culture with S. aureus showed between 2 and 1-log₁₀ (90%) reduction. In the following culture, a significant decrease in efficiency is detectable with 1-log reduction only with 1 mg.mL⁻¹. Fmoc-FFY hydrogel coating, self-assembled with 1 mg.mL⁻¹ Fmoc-FFpY, showed only 1-log reduction of bacterial load against Gram-negative E. coli after 24 h with no significant inhibition at the second culture (Figure 3C). A selective antibacterial effect of Fmoc-FFY hydrogel coating was

observed against S. aureus Gram-positive bacteria in contrast to E. coli Gram-negative bacteria.

The mechanism of action of Fmoc-FFY hydrogels could be attributed to a combination of two factors: the fibrillary β-sheet structure of the self-assembly and the peptide hydrophobicity as it was reported for β-amyloid peptides (Kagan et al., 2012; Last and Miranker, 2013). Pioneering works of Moir and coll. demonstrated the antimicrobial activity of Alzheimer's β-amyloid peptides *in vitro* and *in vivo* (Soscia et al., 2010; Kumar et al., 2016). Diphenylalanine moieties (FF), identified as the core recognition motif of β-amyloid peptides, were reported to self-assemble in discrete and stiff nanotubes adopting β-sheet structures (Reches and Gazit, 2003). Self-assembled FF peptides have the ability to bind to the surface of bacterial membrane, to aggregate and to orient themselves to optimize hydrophilic/hydrophobic interactions, inducing a surface tension leading to membrane depolarization, pore formation and release of cellular content (Schnaider et al., 2017). Moreover, FF peptides also induce upregulation of stress response regulons at sub-lethal concentration causing severe damage to bacterial morphology. Recently, the antibacterial activity of self-assembled Fmoc-F was attributed to the peptide release from the hydrogel inducing oxidative and osmotic stress as well as altering bacterial membrane integrity (Gahane et al., 2018). The comparison of Fmoc-FF cationic peptide, designed with a pyridinium moiety at the C-terminal, with other Fmoc-cationic peptides showed that the proximity of fluorenyl and phenyl moieties achieves an optimum hydrophobicity environment improving the antibacterial activity (Debnath et al., 2010). In summary, the antibacterial property of the Fmoc-FFY hydrogel coating could be explained by the ability of the β-sheet self-assembled nanofibers to bind and aggregate on the surface of the bacterial membrane, altering the membrane integrity and also to oxidative and osmotic stresses leading to the death of the bacteria. This mechanism is confirmed by the efficiency of peptide self-assembly at low concentration in comparison to the non-self-assembled peptide. In contrast to Gram-positive, Gram-negative bacteria have an additional outer bilayer membrane, formed by lipopolysaccharides and phospholipids, that makes difficult the diffusion of molecules across the membrane and renders them more resistant to break (Haldar et al., 2005). The cytotoxicity of the Fmoc-FFY gel was determined *in vitro*, through a MTT indirect test, employing NIH 3T3 mouse embryonic fibroblasts
<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

**FIGURE 3** | Colony-forming unit (CFU) log₁₀ reduction on planktonic (A) *S. aureus* cells incubated in the presence of different concentrations of Fmoc-FFpY and of Fmoc-FFY hydrogel, self-assembled with different concentrations of Fmoc-FFpY, (B) *S. aureus* cells incubated on Fmoc-FFY hydrogel coatings, self-assembled with different concentrations of Fmoc-FFpY on NPs@AP coating and (C) *E. coli* cells incubated on Fmoc-FFY hydrogel coating, self-assembled with 1 mg.mL⁻¹ Fmoc-FFpY solution on NPs@AP coating, as a function of the number of repetitive culture. The coating was brought in contact with a fresh pathogen suspension for 24 h. In repetitive culture experiments, the supernatant was removed and replaced every 24 h by a fresh suspension. (D) Cytotoxicity assay of Fmoc-FFY gel through MTT indirect test using NIH 3T3 mouse embryonic fibroblasts cells. Uncoated glass substrates were employed as controls (CTRL). Diagrams include the results from three independent experiments in triplicate and results are shown as mean ± s.e.m. and the ANOVA results at a significance level of **p** < 0.01 vs. uncoated glass (negative control, CTRL).

cells (Figure 3D). NIH 3T3 cell viability do not show any decrease of up to 48 h proving that Fmoc-FFY gel is not cytotoxic. This result confirmed the absence of toxicity of Fmoc-FF peptides when they are in self-assembled form (Truong et al., 2015). Besides, Fmoc-FF peptides have been used as 3-D cell culture scaffolds for chondrocytes (Jayawarna et al., 2006) or astrocytes (Liebmann et al., 2007).

CONCLUSION

In this work a phosphorylated peptide, Fmoc-FFpY, able to be
solubilized in water at room temperature, was transformed in

Fmoc-FFY hydrogelator by action of NPs@AP immobilized on
a surface. The hydrogel coating, based on fibers homogeneously
distributed all around the sample, was efficient to prevent
S. aureus growth with 4-log₁₀ of viable bacteria. This coating
could serve in infections were Gram positive bacteria are
prevalent, e.g., intravascular catheter infections. This kind of
antibacterial self-assembled hydrogel represents a very simple
and efficient method to coat biomaterial surfaces for protection
against bacteria proliferation. In clinical practice to ensure
the integrity of the hydrogel coating, one idea would be to
trigger the peptide self-assembly *in situ* after implantation of
the medical device by addition of the peptide solution near the
enzyme functionalized implant surface. With the prospect of
long-term use of the antibacterial coating, the surface triggered self-assembly of peptide could be done on porous surfaces or even inside the pores of foams. The high surface of coverage, obtained on rough or porous implants, could lead to sustained antibacterial property as the coating maintained its property even after being in contact several times with fresh inoculated *S. aureus* suspension. Moreover in a curative strategy, the peptide self-assembly could be triggered by using enzymes naturally secreted by bacteria to obtain the antibacterial gel either inside the cells or at their surface using overexpression of phosphatase by bacteria (Yang et al., 2007) or by mammalian cancer cells (Pires et al., 2015).

## DATA AVAILABILITY STATEMENT

All datasets presented in this study are included in the article/Supplementary Material.

## AUTHOR CONTRIBUTIONS

PS and FB conceived the project. MC-G, LJ, PS, and FB designed the experiments. MC-G carried out the preparation of all samples under study, QCM-D analyses, gelation tests, circular dichroism, and fluorescence spectroscopy characterization. MI performed

the AFM characterization and bacteria tests. MC-G, MI, and FB contributed to the interpretation of the results. AC and MS carried out the SEM and cryo-SEM experiments. All authors contributed to the article and approved the submitted version.

## FUNDING

MI thanks the Higher Education Commission (HEC) Pakistan for his Ph.D. scholarship. We gratefully acknowledge the financial support from the Agence Nationale de la Recherche (EASA, ANR-18-CE06-0025-03).

## ACKNOWLEDGMENTS

ICS microscopy and characterization platforms are acknowledged for the use of the SEM and the fluorescence spectroscopy, respectively.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2020.00938/full#supplementary-material

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Criado-Gonzalez, Iqbal, Carvalho, Schmutz, Jierry, Schaaf and Boulmedais. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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# Evaluating the Toxicity of Ionic Liquids on Shewanella sp. for Designing Sustainable Bioprocesses

Hakima Kebaili¹,²*, Antonia Pérez de los Ríos¹, María José Salar-García³*,
Víctor Manuel Ortiz-Martínez³*, Mostefa Kameche², Jesús Hernández-Fernández¹ and
Francisco J. Hernández-Fernández¹*

¹Department of Chemical Engineering, Faculty of Chemistry, University of Murcia (UMU), Murcia, Spain, ²Laboratory of Physico-Chemistry of Materials, Catalysis and Environment, Faculty of Chemistry, University of Sciences and Technology of Oran-Mohamed Boudiaf, Oran, Algeria, ³Department of Chemical and Environmental Engineering, Technical University of Cartagena (UPCT), Campus La Muralla, Cartagena, Spain

## OPEN ACCESS

**Edited by:**
Emilia Morallon,
University of Alicante, Spain

**Reviewed by:**
Adil Denizli,
Hacettepe University, Turkey
Guillermo Díaz-Sainz,
University of Cantabria, Spain

**\*Correspondence:**
María José Salar-García
mariajose.salar@upct.es
Francisco J. Hernández-Fernández
fjhernan@um.es
Hakima Kebaili
kebaillhakima@gmail.com
Victor Manuel Ortiz-Martínez
victor.ortiz@upct.es

**Specialty section:**
This article was submitted to
Biomaterials,
a section of the journal
Frontiers in Materials

**Received:** 01 July 2020
**Accepted:** 13 October 2020
**Published:** 27 November 2020

**Citation:**
Kebaili H, Pérez de los Ríos A, Salar-García MJ, Ortiz-Martínez VM, Kameche M, Hernández-Fernández J and Hernández-Fernández FJ (2020) *Evaluating the Toxicity of Ionic Liquids on Shewanella sp. for Designing Sustainable Bioprocesses*. *Front. Mater.* 7:578411. doi: 10.3389/fmats.2020.578411

Ionic liquids (ILs) are widely used as reaction and separation media in many technologies due to their unique and advantageous physicochemical properties. Thus, further studies approaching the study of the toxicity of these materials are required. Moreover, they are utilized in devices in which microorganisms such as *Shewanella* sp. act as biocatalysts. Thus, in this work, the toxicity of 69 ILs on the marine bacterium *Shewanella* sp. was tested. Specifically, the ILs analyzed were based on the cations imidazolium, pyridinium, pyrrolidinium, piperidinium, morpholinium, oxazolinium, phosphonium, ammonium, and sulfonium, in combination with different anions. The toxicities of this wide group of ILs on *Shewanella* sp. were determined using two methods: 1) the agar disk-diffusion test and 2) the growth inhibition test in liquid media. The relationship between toxicity and IL chemical structure was elucidated. A decrease in the hydrophobicity of the alkyl chain length was found to be a key factor to reduce IL toxicity. On the other hand, phosphonium-based ILs containing long alkyl chains were shown to be largely incompatible with *Shewanella* sp.

**Keywords:** ionic liquids, toxicity, *Shewanella* sp, sustainable materials, biocompatibility

## HIGHLIGHTS

* ILs as clean and green alternatives to organic solvents in bioprocesses

* Screening IL toxicity toward *Shewanella* sp. for bioprocess applications

* Analysis of chemical structure contributions to IL toxicity toward *Shewanella* sp.

* Tool for the adequate cation/anion combination to synthesis nontoxic ILs

## INTRODUCTION

The genus *Shewanella* was first described in 1931. This microorganism responsible for butter putrefaction was initially named as *Achromobacter putrefaciens* but after further studies and biochemical characterizations, the species isolated in 1931 was then reclassified into a new genus, *Shewanella* (Derby and Hammer, 1931; MacDonell and Colwell, 1985). So far, all members belonging to this genus are facultatively anaerobic, which allows them to survive in a wide variety of environments, although they are usually found in aquatic or marine habitats (Hau
and Gralnick, 2007). One of the main characteristics of the *Shewanella* genus is its capability to grow at low temperatures. Although most of the strains are psychrotolerant with optimum growth temperature above 16°C, many strains are psychrophilic and can easily grow below this temperature (Hau and Gralnick, 2007). The capability of *Shewanella* to use several external electron acceptors to respire, which enables this genus to live in extreme conditions, as well as the possibility of being genetically modified, has boosted its application in a wide variety of technological fields (Hau and Gralnick, 2007; Marsili et al., 2008; Zou et al., 2019), from bioremediation of polluted environments (Varia et al., 2014; Zhang et al., 2018) and biosensing (Webster et al., 2014) to bioenergy production in microbial fuel cell technology (Jorge and Hazael, 2016; Kumar et al., 2016).

Ionic liquids (ILs) are combinations of cations (i.e., imidazolium, pyridinium, and ammonium) and anions (i.e., hexafluorophosphate, tetrafluoroborate, and bis [(trifluoromethyl)sulfonyl]imide) that remain liquid at temperatures below 100°C. In recent years, these compounds have drawn the attention of the scientific community due to their unique properties such as negligible vapor pressure, chemical and thermal stability, nonflammability, high ionic conductivity, wide electrochemical potential window, and solvation ability, among other features (Ventura et al., 2012b). ILs are also considered clean and green alternatives to organic solvents mainly due to their low vapor pressure, which prevents their release into the atmosphere. Besides, their physical and chemical properties can be adapted to specific applications by the fine-tuning of the cation structure and by selecting proper cation-anion combinations. Since a wide variety of ion combinations are possible, it is of the highest importance to outline rational guidelines to develop technologically suitable but also environmentally harmless ILs (Latała et al., 2009).

The emergence of ILs as solvents has instigated several new and exciting areas of scientific research including organic synthesis (Welton, 1999; Earle et al., 2008), catalysis (Welton, 1999; Pârvulescu and Hardacre, 2007), electrodeposition (Endres, 2002; Zein El Abedin et al., 2006; El Abedin and Endres, 2007), dye-sensitized solar cells (Gorlov and Kloo, 2008), and lubrication (Ye et al., 2001). Moreover, within the field of nanotechnology, ILs have been synthesized with finely tuned nanoscopic structures for the preparation of polymers (Haddleton et al., 2008; Kim et al., 2008), nanoparticles (Dupont et al., 2002; Schrekker et al., 2007), nanotubes (Barisci et al., 2004; Shim and Kim, 2009), and microemulsions (Eastoe et al., 2005; Qiu and Texter, 2008). ILs are currently being investigated worldwide for cutting edge applications, especially for electrochemical devices and energy production such as lithium batteries (Sakaebe et al., 2005; Ishikawa et al., 2006), electrochemical capacitors (Balducci et al., 2005), electrochemical actuators (Ding et al., 2003), and light-emitting electrochemical cells (Yang et al., 2003). In the field of biocatalysis, ILs have been used free of solvent (Toral et al., 2007; de los Ríos et al., 2012; Lee et al., 2015), adsorbed (Hernández et al., 2006; Lee et al., 2007; Jiang et al., 2009; Zou et al., 2014), and covalently linked to particles enzymes (Lozano

et al., 2010; Suo et al., 2018; Xiang et al., 2018) or as polymeric ILs (Nakashima et al., 2009) in enzyme particles, in all cases to create an adequate enzyme microenvironment and improve the catalytic efficiency (Hernández et al., 2006; Toral et al., 2007; Lozano et al., 2010; de los Ríos et al., 2012; Lee et al., 2015). ILs and *Shewanella* sp. have been recently used together in microbial fuel cell technology for bioenergy production (Zhao et al., 2013; Wei et al., 2016) and the biofabrication of nanoparticles (Wang et al., 2018). Due to the enormous range of IL application and the multiple benefits of including bacteria in certain types of bioprocesses, it is crucial to address the biocompatibility of IL-based materials for progress in the design of sustainable processes.

In this context, this work investigates the biocompatibility of ILs containing different cation and anion structures toward *Shewanella* sp. Thus, the toxicity of a total number of 69 ILs based on imidazolium, pyridinium, pyrrolidinium, piperidinium, morpholinium, oxazolinium, phosphonium, ammonium, and sulfonium combined with different anions has been analyzed in solid and liquid media. The results obtained are in-depth discussed and related to the structural characteristic of the ILs, which enables identifying key factors for the design of environmentally friendly ILs with lower ecotoxicological risks than conventional organic solvents.

# MATERIALS AND METHODS

## Ionic Liquids

The ILs investigated in the current work are grouped according to the type of cation present in their structure. Tables 1–8 include the name of each IL analyzed, together with its structure and water solubility. These tables also include the nomenclature used for each IL (abbreviation name). All ILs were supplied by IoLiTec (Ionic Liquids Technologies).

## Culture of *Shewanella* sp.

The strain of *Shewanella* sp. was provided by the Aquatic Ecology Laboratory (University of Murcia, Spain) and kept on Luria-Bertani Agar (LB). A single colony was used to grow the inoculum on LB broth at 25°C with shaking at 200 rpm. All chemicals and media reagents were purchased from Sigma-Aldrich.

## Toxicity Analysis

The toxicity of 69 ILs toward *Shewanella* sp. was analyzed by using two different methods: 1) agar disk-diffusion test and 2) toxicity in liquid media. Both methods are described below.

### Agar Disk-Diffusion Test

The agar disk-diffusion method was used for the first time in 1940 by Heatley (Heatley, 1944) being now the most common technique for determining the antimicrobial properties of many compounds (Balouiri et al., 2016). So far, a wide variety of bacteria has been successfully tested by this method by varying the culture media and the incubation conditions as well as the evaluation criteria for the inhibition zones (Clinical and Laboratory Standards Institute, 2015). This method consists of
<table><caption>TABLE 1 | Imidazolium-based ionic liquids.</caption><thead><tr><th>Full name</th><th>Abbreviation name</th><th>Structure</th><th>Water miscibility</th><th>State 25 °C</th><th>Ref.</th><th>Inhibition zone/cm</th><th>μ/% in LB broth</th></tr></thead><tbody><tr><td>1-(2-Hydroxypropyl)-methylimidazolium chloride</td><td>[HOPmim<sup>+</sup>][Cl<sup>-</sup>]</td><td>CH<sub>3</sub>OH N<sup>+</sup>CH<sub>3</sub>Cl<sup>-</sup></td><td>Soluble</td><td>Liquid</td><td>Pérez De Los Ríos et al., 2017</td><td>0</td><td>73.65 ± 2.69</td></tr><tr><td>1-Butyl-3-methylimidazolium chloride</td><td>[Bmim<sup>+</sup>][Cl<sup>-</sup>]</td><td>CH<sub>3</sub>CH<sub>2</sub>N<sup>+</sup>CH<sub>3</sub>Cl<sup>-</sup></td><td>Soluble</td><td>Solid</td><td>Dharaskar et al., 2013</td><td>0</td><td>30.51 ± 1.29</td></tr><tr><td>1-Hexyl-3-methylimidazolium chloride</td><td>[Hmim<sup>+</sup>][Cl<sup>-</sup>]</td><td>CH<sub>3</sub>CH<sub>2</sub>N<sup>+</sup>CH<sub>3</sub>Cl<sup>-</sup></td><td>Soluble</td><td>Liquid</td><td>Pérez De Los Ríos et al., 2017</td><td>0</td><td>272.95 ± 9.93</td></tr><tr><td>1-Ethyl-3-methylimidazolium tetrafluoroborate</td><td>[Emim<sup>+</sup>][BF<sub>4</sub><sup>-</sup>]</td><td>H<sub>3</sub>CN<sup>+</sup>CH<sub>3</sub>Cl<sup>-</sup></td><td>Soluble</td><td>Liquid</td><td>Pérez De Los Ríos et al., 2017</td><td>0</td><td>40.30 ± 16.53</td></tr><tr><td>1-Butyl-3-methylimidazolium tetrafluoroborate</td><td>[Bmim<sup>+</sup>][BF<sub>4</sub><sup>-</sup>]</td><td>CH<sub>3</sub>CH<sub>2</sub>N<sup>+</sup>CH<sub>3</sub>Cl<sup>-</sup></td><td>Soluble</td><td>Liquid</td><td>Dharaskar et al., 2016b</td><td>0.7 ± 0.6</td><td>0</td></tr><tr><td>1-Methyl-3-octylimidazolium tetrafluoroborate</td><td>[Moim<sup>+</sup>][BF<sub>4</sub><sup>-</sup>]</td><td>CH<sub>3</sub>CH<sub>2</sub>N<sup>+</sup>CH<sub>3</sub>Cl<sup>-</sup></td><td>Insoluble decomposition in the presence of water</td><td>Liquid</td><td>Freire et al., 2007; Galai et al., 2015</td><td>2.6 ± 0.1</td><td>0</td></tr><tr><td>1-Butyl-2,3-dimethylimidazolium tetrafluoroborate</td><td>[BDImim<sup>+</sup>][BF<sub>4</sub><sup>-</sup>]</td><td>CH<sub>3</sub>CH<sub>2</sub>N<sup>+</sup>CH<sub>3</sub>Cl<sup>-</sup></td><td>Soluble</td><td>Liquid</td><td>Pérez De Los Ríos et al., 2017</td><td>0</td><td>64.20 ± 4.59</td></tr><tr><td>1-Methoxymethyl-3-methylimidazolium tetrafluoroborate</td><td>[MOMim<sup>+</sup>][BF<sub>4</sub><sup>-</sup>]</td><td>CH<sub>3</sub>CH<sub>2</sub>N<sup>+</sup>CH<sub>3</sub>Cl<sup>-</sup></td><td>Soluble</td><td>Liquid</td><td>Pérez De Los Ríos et al., 2017</td><td>0</td><td>263.74% ± 6.85</td></tr><tr><td>1-Methoxyethyl-3-methylimidazolium tetrafluoroborate</td><td>[MOEmim<sup>+</sup>][BF<sub>4</sub><sup>-</sup>]</td><td>CH<sub>3</sub>CH<sub>2</sub>N<sup>+</sup>CH<sub>3</sub>Cl<sup>-</sup></td><td>Insoluble</td><td>Liquid</td><td>Pérez De Los Ríos et al., 2017</td><td>0</td><td>34.74 ± 18.82</td></tr><tr><td>1-Butyl-3-methylimidazolium hexafluorophosphate</td><td>[Bmim<sup>+</sup>][PF<sub>6</sub><sup>-</sup>]</td><td>CH<sub>3</sub>CH<sub>2</sub>N<sup>+</sup>CH<sub>3</sub>Cl<sup>-</sup></td><td>Insoluble</td><td>Liquid</td><td>Carda-Broch et al., 2003</td><td>2.9 ± 0.1</td><td>0 (RWM)</td></tr></tbody></table>

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<table><caption>TABLE 1 | (Continued) Imidazolium-based ionic liquids.</caption><thead><tr><th>Full name</th><th>Abbreviation name</th><th>Structure</th><th>Water miscibility</th><th>State 25 °C</th><th>Ref.</th><th>Inhibition zone/cm</th><th>μ/% in LB broth</th></tr></thead><tbody><tr><td>1-Methyl-3-octylimidazolium hexafluorophosphate</td><td>[Omim<sup>+</sup>][PF<sub>6</sub><sup>-</sup>]</td><td></td><td>Insoluble decomposition in the presence of water</td><td>Liquid</td><td>Freire et al., 2007; Iolitec, 2014; Galai et al., 2015</td><td>2.6 ± 0.2</td><td>24.19% ± 6.25</td></tr><tr><td>1-Methoxymethyl-3-methylimidazolium hexafluorophosphate</td><td>[MOMmim<sup>+</sup>][PF<sub>6</sub><sup>-</sup>]</td><td></td><td>Soluble</td><td>Solid</td><td>Galai et al., 2015</td><td>1.5 ± 0.0</td><td>24.16 ± 4.37</td></tr><tr><td>1-(Ethoxyethyl)-3-methylimidazolium hexafluorophosphate</td><td>[EOEmim<sup>+</sup>][PF<sub>6</sub><sup>-</sup>]</td><td></td><td>Soluble</td><td>Solid</td><td>Pérez De Los Ríos et al., 2017</td><td>2.0 ± 0.4</td><td>45.92 ± 14.12</td></tr><tr><td>1-Butyl-3-methylimidazolium nitrate</td><td>[Bmim<sup>+</sup>][NO<sub>3</sub><sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Pérez De Los Ríos et al., 2017</td><td>0</td><td>102.80 ± 3.46</td></tr><tr><td>1-(2-Hydroxypropyl)-3-methylimidazolium nitrate</td><td>[HOPmim<sup>+</sup>][NO<sub>3</sub><sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Pérez De Los Ríos et al., 2017</td><td>0.5 ± 0.0</td><td>0</td></tr><tr><td>1-Butyl-3-methylimidazolium acetate</td><td>[Bmim<sup>+</sup>][CH<sub>3</sub>COO<sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Pérez De Los Ríos et al., 2017</td><td>0.8 ± 0.0</td><td>0</td></tr><tr><td>1-Butyl-3-methylimidazolium glycolate</td><td>[Bmim<sup>+</sup>][CH<sub>3</sub>OHCOO<sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Pérez De Los Ríos et al., 2017</td><td>0.5 ± 0.0</td><td>30.17 ± 8.57</td></tr><tr><td>1-Butyl-3-methylimidazolium methylcarbonate</td><td>[Bmim<sup>+</sup>][MeCOO<sub>2</sub><sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Galai et al., 2015</td><td>1.5 ± 0.4</td><td>0</td></tr><tr><td>1-Butyl-3-methylimidazolium thiocyanate</td><td>[Bmim<sup>+</sup>][SCN<sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Dharaskar et al., 2016a</td><td>1.5 ± 0.0</td><td>0</td></tr><tr><td>1-Ethyl-3-methylimidazolium dicyanamide</td><td>[Emim<sup>+</sup>][dca<sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Pérez De Los Ríos et al., 2017</td><td>0</td><td>24.13 ± 11.44</td></tr></tbody></table>

(Continued on following page)
<table><caption>TABLE 1 | (Continued) Imidazolium-based ionic liquids.</caption><thead><tr><th>Full name</th><th>Abbreviation name</th><th>Structure</th><th>Water miscibility</th><th>State 25 °C</th><th>Ref.</th><th>Inhibition zone/cm</th><th>μ/% in LB broth</th></tr></thead><tbody><tr><td>1-Ethyl-3-methylimidazolium bis(trifluoromethyl) sulfonyl)imide</td><td>[Emim<sup>+</sup>][NTf<sub>2</sub>]</td><td>[Image of structure]</td><td>Insoluble</td><td>Liquid</td><td>Zhou et al., 2012</td><td>0.7 ± 0.3</td><td>0</td></tr><tr><td>1-Butyl-3-methylimidazolium bis(trifluoromethyl) sulfonyl)imide</td><td>[Bmim<sup>+</sup>][NTf<sub>2</sub>]</td><td>[Image of structure]</td><td>Insoluble</td><td>Liquid</td><td>Papaiconomou et al., 2006</td><td>1.0 ± 0.1</td><td>0</td></tr><tr><td>1-Methyl-3-octylimidazolium bis(trifluoromethyl) sulfonyl)imide</td><td>[Omim<sup>+</sup>][NTf<sub>2</sub>]</td><td>[Image of structure]</td><td>Insoluble</td><td>Liquid</td><td>Papaiconomou et al., 2006</td><td>2.3 ± 0.4</td><td>0</td></tr></tbody></table>

inoculating agar plates with a standardized inoculum of a specific microorganism. Then, the test compound embedded in small pieces of filter paper was placed on the agar surface and the Petri dishes were incubated under suitable conditions for the growth of the microorganisms. Finally, the antimicrobial activity of the test compound was evaluated according to the diameter of the inhibition growth zone. Although this method is not entirely satisfactory to determine the minimum inhibitory concentration (MIC), it brings many advantages over other techniques such as its simplicity and suitability to assess the compatibility between a wide variety of compounds and microorganisms as well as its low cost (Balouiri et al., 2016).

Due to the great interest of the scientific community in the search for new and safe applications of ILs, the agar disk-diffusion method has also been used to determine the biocompatibility of these green solvents (Rebros et al., 2009; Ventura et al., 2012a). Thus, this procedure was selected to evaluate the inhibitory capacity of a wide variety of ILs on the growth of *Shewanella* sp. To this end, 100 µl of *Shewanella* sp. culture incubated overnight was spread on an LB agar plate. Each IL was added to sterile, preweighted disks of filter paper (around 6 mm of diameter), and then placed on the surface of the Petri agar dish. ILs in solid-state were weighed and then sterile-distilled water was added dropwise until the salt was completely dissolved. An aliquot of 5 µl of the salt solution was added to the disk of filter paper and the amount embedded ranged between 0.004 and 0.0125 g. All Petri dishes were incubated overnight in a static incubator at 25°C, and the radius of the inhibition zone around the filter paper was measured according to Rebros et al. (2009). Each IL was tested in triplicate and the inhibition zones reported in the present work are the average of the three replicates unless otherwise stated.

## Toxicity in Liquid Media

To delve into the biocompatibility of the ILs with *Shewanella* sp., an alternative method based on measuring the growth rates in LB broth medium containing yeast extract, peptone, glucose, and NaCl in the presence and absence of IL was used. In order to decrease the viscosity, water-miscible ILs were weighed and mixed with sterile-distilled water to produce a stock solution (95% w/v). Viscous ILs along with those with unusual phase behavior were also prepared in the same way. The stock solutions were vortexed before adding an aliquot of 40 µl to the microcultures up to a final concentration of 2% (v/v). Nonviscous water-immiscible ILs were added directly to the cultures by volumetric measurement to produce a biphasic system 2% v/v. The cultures were inoculated with 100 µl from an overnight culture of *Shewanella* sp. grown in the same medium. The flasks were incubated for 24 h at 25°C with continuous and intensive shaking.

The optical density (OD) of each culture was measured every 2 h and the growth curve was determined for each case. The specific growth rate (μ, h⁻¹) was calculated by selecting two points at times t₁ and t₂ in the exponential growth phase and applying the following equation:

$$ \mu = \frac{\text{In} (\text{OD}_2/\text{OD}_1)}{t_1 - t_2}. $$
<table><caption>TABLE 2 | Pyridinium-based ionic liquids.</caption><thead><tr><th>Full name</th><th>Abbreviation name</th><th>Structure</th><th>Water miscibility</th><th>State 25°C</th><th>Ref.</th><th>Inhibition zone/cm</th><th>μ/% in LB broth</th></tr></thead><tbody><tr><td>1-Butyl-3-methylpyridinium tetrafluoroborate</td><td>[BMPy<sup>+</sup>][BF<sub>4</sub><sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Ortega et al., 2007; Galai et al., 2015</td><td>0</td><td>232.69 ± 4.22</td></tr><tr><td>1-Butyl-3-methylpyridinium dicyanamide</td><td>[BMPy<sup>+</sup>][dca<sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Bandrés et al. 2008; Kalčíková et al., 2012; Galai et al., 2015</td><td>1.0 ± 0.0</td><td>155.18 ± 34.40</td></tr><tr><td>1-Methyl-3-octylpyridinium dicyanamide</td><td>[MOPy<sup>+</sup>][dca<sup>-</sup>]</td><td></td><td>Insoluble</td><td>Liquid</td><td>Galai et al., 2015</td><td>1.1 ± 0.0</td><td>0</td></tr><tr><td>1-Methylpyridinium methylsulfate</td><td>[MPy<sup>+</sup>][MeSO<sub>4</sub><sup>-</sup>]</td><td></td><td>Soluble</td><td>Solid</td><td>Galai et al., 2015</td><td>0</td><td>170.77 ± 9.85</td></tr><tr><td>1,4 dimethyl pyridinium methylsulfate</td><td>[MMPy<sup>+</sup>][Me SO<sub>4</sub><sup>-</sup>]</td><td></td><td>Soluble</td><td>Solid</td><td>Galai et al., 2015</td><td>1.8 ± 0.4</td><td>317.77 ± 9.99</td></tr><tr><td>1-Ethylpyridinium ethyl sulfate</td><td>[EPy<sup>+</sup>][EtSO<sub>4</sub><sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Benito et al., 2014; Galai et al., 2015</td><td>0</td><td>286.16 ± 10.19</td></tr><tr><td>1-Ethylpyridinium bis [(trifluoromethyl)sulfonyl]imide</td><td>[EPy<sup>+</sup>][NTf<sub>2</sub><sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Benito et al., 2014; Galai et al., 2015; Iolitec, 2015b</td><td>0.9 ± 0.0</td><td>19.68 ± 5.83</td></tr></tbody></table>
<table><caption>TABLE 3 | Pyrrolidinium-based ionic liquids.</caption><thead><tr><th>Full name</th><th>Abbreviation name</th><th>Structure</th><th>Water miscibility</th><th>State 25 °C</th><th>Ref.</th><th>Inhibition zone/cm</th><th>μ/% in LB broth</th></tr></thead><tbody><tr><td>1-Butyl-1-methyl pyrrolidinium chloride</td><td>[BMPyr<sup>+</sup>][Cl<sup>-</sup>]</td><td>H<sub>3</sub>C<sup>+</sup>N<sup>-</sup>CH<sub>3</sub>Cl<sup>-</sup></td><td>Soluble</td><td>Solid</td><td>Galai et al., 2015; Iolitec, 2015a</td><td>0.9 ± 0.0</td><td>0</td></tr><tr><td>1-Butyl-1-methyl pyrrolidinium hexafluorophosphate</td><td>[BMPyr<sup>+</sup>][PF<sub>6</sub><sup>-</sup>]</td><td>H<sub>3</sub>C<sup>+</sup>N<sup>-</sup>CH<sub>3</sub>F<sup>-</sup>F<sup>-</sup>F<sup>-</sup>F<sup>-</sup></td><td>Insoluble</td><td>Solid</td><td>Iolitec, 2011c; Ventura et al., 2013</td><td>0.8 ± 0.0</td><td>0</td></tr><tr><td>1-Butyl-1-methyl pyrrolidinium dicyanamide</td><td>[BMPyr<sup>+</sup>][dca<sup>-</sup>]</td><td>H<sub>3</sub>C<sup>+</sup>N<sup>-</sup>CH<sub>3</sub>N=N<sup>-</sup>N=N<sup>-</sup></td><td>Soluble</td><td>Liquid</td><td>Iolitec, 2011b; Cumicheo et al., 2015</td><td>0</td><td>88.78 ± 14.74</td></tr><tr><td>1-Butyl-1-methyl pyrrolidinium triflate</td><td>[BMPyr<sup>+</sup>][TFO<sup>-</sup>]</td><td>H<sub>3</sub>C<sup>+</sup>N<sup>-</sup>CH<sub>3</sub>O<sup>-</sup>S<sup>-</sup>S<sup>-</sup>F<sup>-</sup>F<sup>-</sup></td><td>Soluble</td><td>Liquid</td><td>Iolitec, 2012b; Fadeeva et al., 2015</td><td>0</td><td>106.33 ± 4.44</td></tr><tr><td>1-Ethyl-1-methyl pyrrolidinium bis[(trifluoromethyl)sulfonyl]imide</td><td>[EMPyr<sup>+</sup>][NTf<sub>2</sub><sup>-</sup>]</td><td>H<sub>3</sub>C<sup>+</sup>N<sup>-</sup>CH<sub>3</sub>F<sup>-</sup>S<sup>-</sup>S<sup>-</sup>F<sup>-</sup>F<sup>-</sup></td><td>Insoluble</td><td>Solid</td><td>Galai et al., 2015; Iolitect, 2011</td><td>1.3 ± 0.3</td><td>0</td></tr><tr><td>1-Butyl-1-methyl pyrrolidinium bis[(trifluoromethyl)sulfonyl]imide</td><td>[BMPyr<sup>+</sup>][NTf<sub>2</sub><sup>-</sup>]</td><td>H<sub>3</sub>C<sup>+</sup>N<sup>-</sup>CH<sub>3</sub>F<sup>-</sup>S<sup>-</sup>S<sup>-</sup>F<sup>-</sup>F<sup>-</sup></td><td>Insoluble</td><td>Liquid</td><td>Iolitec, 2011a; Fadeeva et al., 2015</td><td>2.2 ± 1.2</td><td>14.33 ± 6.31</td></tr></tbody></table>
<table><caption>**TABLE 4** | Piperidinium-based ionic liquids.</caption><thead><tr><th>Full name</th><th>Abbreviation name</th><th>Structure</th><th>Water miscibility</th><th>State at 25 °C</th><th>Ref.</th><th>Inhibition zone/cm</th><th>μ/% in LB broth</th></tr></thead><tbody><tr><td>1-Butyl-1-methylpiperidinium bis[(trifluoromethyl)sulfonyl] imide</td><td>[BMPi<sup>+</sup>][NTf<sub>2</sub><sup>-</sup>]</td><td><img src="chemical_structure.png" alt="Chemical structure of 1-Butyl-1-methylpiperidinium bis[(trifluoromethyl)sulfonyl] imide"></td><td>Insoluble</td><td>Liquid</td><td>Iolitec, 2012a; Galai et al., 2015</td><td>1.6 ± 0.1</td><td>0</td></tr></tbody></table>

<figure><img src="image_26.png" /><figcaption>**TABLE 5** | Morpholinium-based ionic liquids.</figcaption></figure>

<table><thead><tr><th>Full name</th><th>Abbreviation name</th><th>Structure</th><th>Water miscibility</th><th>State at 25 °C</th><th>Ref.</th><th>Inhibition zone/cm</th><th>μ/% in LB broth</th></tr></thead><tbody><tr><td>Ethyl methyl morpholinium dicyanamide</td><td>[EMMOR<sup>+</sup>][dca<sup>-</sup>]</td><td><img src="chemical_structure.png" alt="Chemical structure of ethyl methyl morpholinium dicyanamide"></td><td>Soluble</td><td>Liquid</td><td>Russina et al., 2013; Peréz de los Ríos et al., 2017</td><td>0</td><td>3.95 ± 1.72</td></tr></tbody></table>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>**TABLE 6** | Oxazolinium-based ionic liquids.</figcaption></figure>

<table><thead><tr><th>Full name</th><th>Abbreviation name</th><th>Structure</th><th>Water miscibility</th><th>State at 25 °C</th><th>Ref.</th><th>Inhibition zone/cm</th><th>μ/% in LB broth</th></tr></thead><tbody><tr><td>Methyloxazolinium methylsulfate</td><td>[Moxa<sup>+</sup>][MeSO<sub>4</sub><sup>-</sup>]</td><td><img src="chemical_structure.png" alt="Chemical structure of methyloxazolinium methylsulfate"></td><td>Soluble</td><td>Liquid</td><td>Galai et al., 2015; Peréz de los Ríos et al., 2017</td><td>1.1 ± 0.3</td><td>25.35 ± 2.79</td></tr></tbody></table>

<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /></figure>
<table><caption>TABLE 8 | Phosphonium-based ionic liquids.</caption><thead><tr><th>Full name</th><th>Abbreviation name</th><th>Structure</th><th>Water miscibility</th><th>State at 25 °C</th><th>Ref.</th><th>Inhibition zone/cm</th><th>μ/% in LB broth</th></tr></thead><tbody><tr><td>Tetradecyl(trihexyl)phosphonium chloride</td><td>[P<sub>6,6,6,14</sub><sup>+</sup>][Cl<sup>-</sup>]</td><td>H<sub>13</sub>C<sub>6</sub>-P-C<sub>6</sub>H<sub>13</sub>H<sub>13</sub>C<sub>6</sub>-P-C<sub>6</sub>H<sub>13</sub>Cl<sup>-</sup></td><td>Insoluble</td><td>Liquid</td><td>Iolitec, 2011g; Galai et al., 2015</td><td>1.0 ± 0.0</td><td>27.72 ± 3.06</td></tr><tr><td>Tetradecyl(trihexyl)phosphonium bromide</td><td>[P<sub>6,6,6,14</sub><sup>+</sup>][Br<sup>-</sup>]</td><td>H<sub>13</sub>C<sub>6</sub>-P-C<sub>6</sub>H<sub>13</sub>H<sub>13</sub>C<sub>6</sub>-P-C<sub>6</sub>H<sub>13</sub>Br<sup>-</sup></td><td>Insoluble</td><td>Liquid</td><td>Iolitec, 2011f; Galai et al., 2015</td><td>5.5 ± 2.8</td><td>0</td></tr><tr><td>Tetraoctylphosphonium bromide</td><td>[P<sub>8,8,8,8</sub><sup>+</sup>][Br<sup>-</sup>]</td><td>H<sub>3</sub>C(H<sub>2</sub>C)<sub>6</sub>H<sub>2</sub>C-P-C<sub>6</sub>H<sub>13</sub>H<sub>13</sub>C<sub>6</sub>H<sub>13</sub>Br<sup>-</sup></td><td>Insoluble</td><td>Solid</td><td>Galai et al., 2015; Iolitec, 2015e</td><td>1.0 ± 0.0</td><td>0</td></tr><tr><td>Tetradecyl(trihexyl)phosphonium tetrafluoroborate</td><td>[P<sub>14, 6,6,6</sub><sup>+</sup>][BF<sub>4</sub><sup>-</sup>]</td><td>H<sub>13</sub>C<sub>6</sub>-P-C<sub>6</sub>H<sub>13</sub>H<sub>13</sub>C<sub>6</sub>H<sub>13</sub>F-F-F-F</td><td>Insoluble</td><td>Liquid</td><td>Åkerstedt et al., 2013; Galai et al., 2015</td><td>1.8 ± 0.4</td><td>108.30 ± 20.08</td></tr><tr><td>Tetrabutylphosphonium dibutyl phosphate</td><td>[P<sub>4,4,4,4</sub><sup>+</sup>][Bu<sub>2</sub>Phos<sup>-</sup>]</td><td>H<sub>9</sub>C<sub>4</sub>-P-C<sub>4</sub>H<sub>9</sub>C<sub>4</sub>H<sub>9</sub>O-P-OC<sub>4</sub>H<sub>9</sub>OC<sub>4</sub>H<sub>9</sub></td><td>Soluble</td><td>Solid</td><td>Zhou et al., 2002; Galai et al., 2015</td><td>0.9 ± 0.2</td><td>75.94 ± 7.29</td></tr><tr><td>Trihexyl(tetradecyl)phosphonium bis-2,4,4-(trimethylpentyl)phosphinate</td><td>[P<sub>6,6,6,14</sub><sup>+</sup>][TMPPhos<sup>-</sup>]</td><td>H<sub>13</sub>C<sub>6</sub>-P-C<sub>6</sub>H<sub>13</sub>H<sub>13</sub>C<sub>6</sub>H<sub>13</sub>Cl<sup>-</sup></td><td>Insoluble</td><td>Liquid</td><td>Galai et al., 2015; Iolitec, 2018</td><td>2.6 ± 0.1</td><td>0</td></tr><tr><td>Trihexyl (tetradecyl)phosphonium decanoate</td><td>[P<sub>6,6,6,14</sub><sup>+</sup>][C<sub>9</sub>COO<sup>-</sup>]</td><td>H<sub>13</sub>C<sub>6</sub>-P-C<sub>6</sub>H<sub>13</sub>H<sub>13</sub>C<sub>6</sub>H<sub>13</sub>Cl<sup>-</sup></td><td>Insoluble</td><td>Liquid</td><td>Iolitec, 2013b; Galai et al., 2015</td><td>1.5 ± 0.2</td><td>0</td></tr><tr><td>Tetradecyl (trihexyl)phosphonium dicyanamide</td><td>[P<sub>6,6,6,14</sub><sup>+</sup>][dca<sup>-</sup>]</td><td>H<sub>13</sub>C<sub>6</sub>-P-C<sub>6</sub>H<sub>13</sub>H<sub>13</sub>C<sub>6</sub>H<sub>13</sub>N-N-N-N</td><td>Insoluble</td><td>Liquid</td><td>Iolitec, 2011h; Galai et al., 2015</td><td>0.8 ± 0.1</td><td>32.28 ± 1.92</td></tr><tr><td>Tributylmethylphosphonium methylsulphate</td><td>[P<sub>4,4,4,1</sub><sup>+</sup>][CH<sub>3</sub>SO<sub>4</sub><sup>-</sup>]</td><td>H<sub>9</sub>C<sub>4</sub>-P-C<sub>4</sub>H<sub>9</sub>C<sub>4</sub>H<sub>9</sub>O-S-S-O-CH<sub>3</sub></td><td>Soluble</td><td>Solid</td><td>Iolitec, 2013a; Galai et al., 2015</td><td>1.0 ± 0.0</td><td>0</td></tr><tr><td>Trihexyl (tetradecyl)phosphonium bis [trifluoromethyl] sulfonyl]imide</td><td>[P<sub>6,6,6,14</sub><sup>+</sup>][NTf<sub>2</sub><sup>-</sup>]</td><td>H<sub>13</sub>C<sub>6</sub>-P-C<sub>6</sub>H<sub>13</sub>H<sub>13</sub>C<sub>6</sub>H<sub>13</sub>Cl<sup>-</sup></td><td>Insoluble</td><td>Liquid</td><td>Iolitec, 2011e; Galai et al., 2015</td><td>1.2 ± 0.2</td><td>0</td></tr></tbody></table>

(Continued on following page)
<table><caption>TABLE 8 | (Continued) Phosphonium-based ionic liquids.</caption><thead><tr><th>Full name</th><th>Abbreviation name</th><th>Structure</th><th>Water miscibility</th><th>State at 25 °C</th><th>Ref.</th><th>Inhibition zone/cm</th><th>μ/% in LB broth</th></tr></thead><tbody><tr><td>Trisobutyl (methyl)phosphonium tosylate</td><td>[P(4,4,4')<sup>+</sup>][TOS<sup>-</sup>]</td><td></td><td>Soluble</td><td>Liquid</td><td>Iolitec, 2011; Galai et al., 2015</td><td>1.0 ± 0.1</td><td>0</td></tr><tr><td>Tributyltetradecylphosphonium dodecylbenzenesulfonate</td><td>[C<sub>6</sub>H<sub>5</sub>]<sup>+</sup>[C<sub>14</sub>H<sub>25</sub>]<sup>P<sup>+</sup></sup><br/>[C<sub>12</sub>H<sub>25</sub>C<sub>6</sub>H<sub>5</sub>SO<sub>3</sub>]</td><td></td><td>Insoluble</td><td>Liquid</td><td>Iolitec, 2011d; Galai et al., 2015</td><td>2.3 ± 0.4</td><td>0</td></tr></tbody></table>

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

This parameter is shown as a percentage of the growth rate in control cultures without ILs. The specific growth rates in % (μ) are reported as a percentage of the growth rate in control cultures grown without ILs (μ₀ = 0.13 h⁻¹) applying the following equation:

$$ \mu(\%) = (\mu^*100)/\mu_0. $$

The specific growth rates reported are the mean of triplicate, unless otherwise stated.

## RESULTS AND DISCUSSION

As preliminary information, three types of inhibition were observed in the presence of ILs. The first type corresponds to symmetric inhibition when the IL diffused correctly in the agar-agar (Figure 1A). In other cases, the asymmetric inhibition zones offer a variable zone of clearance, rather than a uniform circle around the filter paper, which can be attributed to nonuniform diffusion (Figure 1B) (Rebros et al., 2009). Finally, there are several ILs that do not form an inhibition zone (Figure 1C). The results obtained with the agar diffusion test were compared with the toxicity test in liquid media. As an example, Figure 2 shows the effect of different ILs on the growth of *Shewanella* sp. The strain was grown in LB broth with and without the respective ILs (2% v/v). The results show that the three ILs displayed [Chol⁺][H₂PO₄⁻], [Bmim⁺][Cl⁻], and [BMPyr⁺][TFO⁻] are not toxic toward the strain. Moreover, stimulation of bacterial growth (the bacteria utilize the ILs as growth factor) can be obtained. Uninoculated controls with IL are also presented. The means of three replicates are shown with standard deviation error bars.

The effect of the ILs on pH change was checked in cultures and inoculated controls using a universal indicator (20 ml) added before and after growth. In general, the influence on pH was small (changes of < 0.5 pH units). On the other hand, as can be expected, the toxicity effect is variable and greatly depends on the chemical structure of each IL. Tables 1–8 include the ILs studied and are organized by type of cation. The structure of the IL and its water solubility, the inhibition zone, and the specific growth rate (μ, h⁻¹) are also presented. The influence of the alkyl substituent of the cation and the type of the cation and anion are systematically analyzed below with the aim to establish toxicity-structure relationships.

### Influence of the Alkyl Substituent of the Cation of Ionic Liquid on Toxicity Toward *Shewanella* sp.

Although there are no data available in the literature related to the toxicity of ILs toward *Shewanella* sp., several works have shown that, for other microorganisms, the toxicity is directly correlated with the chain length of the alkyl substituent in the cation (Couling et al., 2006; Luis et al., 2007; Romero et al., 2008; Pretti et al., 2009; Pérez De Los Ríos et al., 2017). The influence of an increasing chain length of R1 or R2 in the imidazolium cation moiety on the cytotoxicity in marine bacteria and two types of
<figure><img src="image_33.png" /><figcaption>**FIGURE 1** | (A) Symmetric inhibition ([C₄mim⁺][PF₆⁻]), (B) asymmetric inhibition ([N₈,₈,₁⁺][NTf₂⁻]), and (C) no inhibition zone ([C₂mim⁺][NTf₂⁻]) in the agar diffusion test.</figcaption></figure>

mammalian cell cultures was also evident in HeLa cells (Stepnowski et al., 2004; Ranke et al., 2007). This effect is currently known as “side-chain effect” (Matzke et al., 2010). For highly lipophilic cations, cytotoxicity does not significantly increase with lipophilicity anymore. It is well known that lipophilicity relationships with biological activity are only linear over a restricted range (Pernak et al., 2003). For *Shewanella* sp., we also found that behavior. In the case of imidazolium cation combined with dicyanamide, a decrease of the specific growth rate and/or an increase in the inhibition radius was found for the series [Emim⁺][dca⁻], [Bmim⁺][dca⁻], [Hmim⁺][dca⁻], and [Omim⁺][dca⁻] and furthermore from [MOMmim⁺][dca⁻] to [MOEmim⁺][dca⁻]. Increasing inhibition radii as alkyl chain length rises were also obtained in the series [Emim⁺][NTf₂⁻], [Bmim⁺][NTf₂⁻], and [Hmim⁺][NTf₂⁻]. Decreasing specific growth rates with the alkyl chain length was observed from [MOMmim⁺][BF₄⁻] to [MOEmim⁺][BF₄⁻]. For this anion, increasing inhibition radii were found from [Bmim⁺][BF₄⁻] to [Omim⁺][BF₄⁻]. According to these results, a higher hydrophobic character in ILs would increase the possibility of their interaction with cell membrane phospholipid bilayers and the hydrophobic domains of the membrane proteins, leading to the disruption of the membrane physiological functions and, consequently, to cell death (Stepnowski et al., 2004; Latała et al., 2005; Ranke et al., 2007; Hernández-Fernández et al., 2015).

In some cases, the toxicity of ILs shows a slight decrease with the increase in alkyl chain length, for instance, from [Bmim⁺][Cl⁻] to [Hmim⁺][Cl⁻] (considering the specific growth rate) and from [Bmim⁺][PF₆⁻] to [Omim⁺][PF₆⁻] (considering both the inhibition radius and the specific growth rate). In this case, an increase in the alkyl chain length also involves a decrease in water media solubility, reducing the effective concentration of the IL in the medium. Besides, the toxic effect of the ILs could depend on the synergy effect of the combined anion and cation and not exclusively on the cation.

As can be observed from **Table 1**, the inclusion of an oxygen atom in the alkyl substituent of the imidazolium ring can significantly decrease the toxicity of imidazolium-based ILs. In fact, it has been observed that [HOPmim⁺][Cl⁻] (μ = 73.5%) displayed much lower toxic effects than [Bmim⁺][Cl⁻] (μ = 30.5%) and also the toxic effect of [MOEmim⁺][BF₄⁻] (μ = 34.7%) on *Shewanella* was lower than that of [Bmim][BF₄⁻] (μ = 0%). This behavior is in agreement with the work by Álvarez-Guerra and Irabien (Álvarez-Guerra and Irabien, 2011), who reported that the presence of oxygenated groups in the structure of cations can lead to a decrease in the ecotoxicity of the IL. In this sense, the presence of oxygen makes the IL more hydrophilic, and therefore, less toxic. On the contrary, the toxicity of [Bmim⁺][NO₃⁻] was higher than the toxicity of [HOPmim][NO₃⁻]. The higher water solubility of [HOPmim⁺][NO₃⁻] and consequently the higher effective concentration in water medium might explain this fact.

## Effect of the Ionic Liquid Cation on Toxicity Toward *Shewanella* sp.

In order to study the effect of the cation, ILs with the same anion and different cation will be compared. This is the best way to systematically study the toxicity of ILs; however, it is important to point out that toxicity synergy effects could occur between anions and cations, and consequently the sequence of toxicity could be modified for different anion families. In this case, it is difficult to separate individual anion and cation contributions to toxicity. In general and in terms of cation toxicity for other microorganisms, ILs containing aromatic cations, such as imidazolium and pyridinium cations, have shown higher levels of toxicity in comparison to those including nonaromatic cations like pyrrolidinium. A higher hydrophobic character of aromatic cations could increase the possibility of interaction with the cell membrane (Stepnowski et al., 2004; Latała et al., 2005; Ranke et al., 2007). Furthermore, the planarity of the cation
<figure><img src="image_34.png" /><figcaption>**FIGURE 2** | (A) Growth profile of *Shewanella* sp. in LB broth. (■) Negative control (LB+ [Chol⁺][H₂PO₄⁻] 2%v/v). (◆) Positive control LB + *Shewanella* sp. (▲) Positive control + [Chol⁺][H₂PO₄⁻]. (B) Growth profile of *Shewanella* sp. in LB broth. (■) Negative control (LB+ [Bmim⁺][Cl⁻] 2%v/v). (◆) Positive control LB + *Shewanella* sp. (▲) Positive control + [Bmim⁺][Cl⁻]. (C) Growth profile of *Shewanella* sp. in LB broth. (■) Negative control (LB+ [BMPyr⁺][TFO⁻] 2%v/v). (◆) Positive control LB + *Shewanella* sp. (▲) Positive control + [BMPyr⁺][TFO⁻].</figcaption></figure>

ring in imidazolium and pyridinium appeared to be also a relevant parameter for increasing IL toxicity, as reported in (Viboud et al., 2012). This fact could be due to the lower steric hindrance of the aromatic cation, which may favor the interactions with the lipid membrane to a greater extent.

For *Shewanella* sp., higher toxicity was found for imidazolium cations compared with pyrrolidinium cations in the case of [Bmim⁺][PF₆⁻] vs. [BMPyr⁺][PF₆⁻] (inhibition radius 2.9 vs. 0.8 cm, respectively), [Bmim⁺][dca⁻] vs. [BMPyr⁺][dca⁻], and

[Bmim][NTf₂⁻] vs. [BMPyr⁺][NTf₂⁻]. No significant differences were found between [Emim⁺][NTf₂⁻] and [BMPyr⁺][NTf₂⁻]. On the contrary, the toxicity of [Bmim⁺][NTf₂⁻] was lower than the toxicity of [BMPyr⁺][NTf₂⁻]. It is also important to remark the higher toxicity observed for [Bmim⁺][BF₄⁻] compared to the toxicity of [BDImim⁺][BF₄⁻]. The inclusion of the methyl substituent in the R2 position of the imidazolium ring could reduce the acid proton in 2, so lowering the toxicity of the IL.

The effect of pyridinium cations was studied by comparison of imidazolium-based ILs with equal alkyl chain length and anions. In all cases, a higher toxicity was found for the imidazolium cation with respect to the pyridinium cation by comparing [Bmim⁻][BF₄⁻] vs. [BMPy⁺][BF₄⁻], [Bmim⁺][dca⁻] vs. [BMPy⁺][dca⁻], and [Omim⁺][dca⁻] vs. [OMPy⁺][dca⁻]. Regarding the piperidinium-based IL studied, [BMPi⁺][NTf₂⁻], there were no significant differences with their homologous imidazolium and pyrrolidinium ones ([Bmim⁺][NTf₂⁻] vs. [BMPyr⁺][NTf₂⁻]). The morpholinium cation in [EMMOR⁺] [dca⁻] can be compared with [Emin⁺][dca⁻]. In both cases, the inhibition radius was zero and the growth rate was higher for the imidazolium-based IL. Matzke et al. (2008) studied the morpholinium compound founding that it only exhibited a moderate effect on the aquatic plant *Lemna minor* and the algae *Scenedesmus vacuolatus*. The oxazolinium cation in [Moxa⁺][MeSO₄⁻] can be compared with the pyridinium cation in [MPy⁺][MeSO₄⁻]; in this case, the toxicity of the pyridinium cation was lower than that of the [Moxa⁺] cation, considering both the inhibition radius (0 vs. 1.1 cm, respectively) and the growth rate (170.7 vs. 25.4%, respectively).

For ILs based on the ammonium cation, different toxicity values were found depending on the alkyl chain length of the cation and the type of the anion. The toxicity values ranged from the highest toxicity for C1EG, C1EG1, and [N₈₈₈₁⁺][Cl⁻] (inhibition radius of 3 cm and 0% growth rate) to the lowest toxicity for [N₈₈₈₁⁺][CF₃SO₃⁻] (1.4 cm of inhibition radius and 179% of growth rate). Wood and Stephens (2010) found that quaternary ammonium salts were generally toxic to *E. coli*. However, it was evidenced that decreasing alkyl chain lengths reduced the toxicity. Our experiment reveals that quaternary ammonium salts are generally toxic, maybe due to their high lipophilic character. However, [Chol⁺][H₂PO₄⁻] and [221PG] (TEGO®-IL-P9) displayed low toxicity toward the strain tested, not inhibiting the bacterial growth in the agar diffusion test. In these cases, the cation contains heteroatoms and short alkyl chains.

Phosphonium-based ILs have been less frequently studied in comparison to other groups despite their high industrial interest (for example, in biotransformation processes such as xenobiotics degradation) (Cieniecka-Rosłonkiewicz et al., 2005). The toxicity results obtained for the phosphonium family suggest that long alkyl chains promote higher toxic effects toward the bacterium (Matzke et al., 2010). The same result was found by Stolte et al. (2008) for phosphonium cations with longer chains (C12, C16, and C18), with a strong inhibitory potential to the used inoculum. The high toxicity of phosphonium halides against *Vibrio fischeri* was also proved (Couling et al., 2006). However, there are possibilities to decrease the antimicrobial activity using long
alkyl chains, e.g., phosphonium based on alkyl chains of 8 and 14 carbons conjugated with the chloride anion. The exchange of the halide by other anions resulted in a loss of antimicrobial activity (Cieniecka-Rosłonkiewicz et al., 2005). In this study, we found that 9 out of 12 phosphonium ILs were very toxic toward *Shewanella* sp. (large radii of inhibition zone and μ of 0% for the bacterial growth in liquid phase). In the case of phosphonium-based IL, the toxicity values ranged from 5.5 cm of inhibition radius and 0% growth rate ([(P<sub>14,6,6,6</sub><sup>+</sup>][Br<sup>-</sup>]) to 1.8 cm of inhibition rate and 108.3% of growth rate ([(P<sub>14,6,6,6</sub><sup>+</sup>][BF<sub>4</sub><sup>-</sup>]). For sulfonium-based ILs, only one compound was assayed, offering high toxicity since no growth of *Shewanella* was observed and the inhibition radius was 1.5 cm.

## Effect of the Ionic Liquid Anion on Toxicity toward *Shewanella* sp.

The effect of the anion composition on the toxicity was analyzed by comparing ILs with different anions and the same cation. As commented above, synergy effects between anion and cation can occur, which makes it difficult to completely isolate individual anion and cation contributions. Several authors have reported that the toxicity is directly correlated to the cation nature, while the anion seems to modulate the toxicity to some extent and in specific cases (Ranke et al., 2004; Couling et al., 2006; Luis et al., 2007; Romero et al., 2008; Pretti et al., 2009). We observed this behavior in several ILs in which the toxicity values were similar when sharing the same cation but containing different anions. For example, for the [Bmim<sup>+</sup>] family, zero growth rate was obtained for the anions [PF<sub>6</sub><sup>-</sup>], [dca<sup>-</sup>], [SCN<sup>-</sup>], [MeCOO<sub>2</sub><sup>-</sup>], [NTf<sub>2</sub><sup>-</sup>], [HSO<sub>4</sub><sup>-</sup>], [CH<sub>3</sub>COO<sup>-</sup>], and [BF<sub>4</sub><sup>-</sup>] with inhibition radii ranging from 2.9 to 0.7 cm. Stolte et al. (2007a) showed that anions with lipophilicity or susceptible to hydrolysis could offer partially drastic effects. In this group, [PF<sub>6</sub><sup>-</sup>] could be included since it can be hydrolyzed in the presence of water, consequently delivering HF to the medium; [NTf<sub>2</sub><sup>-</sup>] could be also included due to its hydrophobicity. Growth rate values higher than zero were obtained for [TFES<sup>-</sup>], [Cl<sup>-</sup>], [CH<sub>2</sub>OHCOO<sup>-</sup>], and [NO<sub>3</sub><sup>-</sup>] (13.6, 30.5, 30, and 102.8, respectively). The [Cl<sup>-</sup>] anion yielded low toxicity when combined with other imidazolium cations such as [HOPmim<sup>-</sup>] and [Hmim<sup>-</sup>]. Wood et al. (2011) tested the toxicity of imidazolium cations combined with halides ([Cl<sup>-</sup>] [Br<sup>-</sup>]) toward *E. coli*. The [Emim<sup>+</sup>] and [Bmim<sup>+</sup>] chlorides and bromides did not produce inhibition zones in the agar diffusion test; however, inhibition zones were observed when the alkyl chain was increased up to six and eight carbons. Short-chained imidazolium salts [EMIM] and [BMIM] with a chloride anion proved weak antimicrobial activity (Łuczak et al., 2010). Therefore, for longer alkyl chain derivatives, the toxicity of the anion can be neglected in comparison to the effects caused by the corresponding cation. On the other hand, for [Cl<sup>-</sup>] combined with pyrrolidinium ([(BMPyr<sup>+</sup>)) and ammonium ([N<sub>8,8,8,1</sub><sup>+</sup>]), high toxicity was found. Cornmell et al. (2008) reported that the IL [N<sub>1,8,8,8</sub><sup>+</sup>][Cl<sup>-</sup>] did not allow any growth for *E. coli*. The [BF<sub>4</sub><sup>-</sup>] anion gave rate growth values over 30% for [Emim<sup>+</sup>] [MOEmim<sup>+</sup>], over 100% for [P<sub>6,6,6,14</sub><sup>+</sup>], and even over 200% for [MOMmim<sup>+</sup>] and [BMPy<sup>+</sup>]; however, nongrowth of *Shewanella*

was observed in [Bmim<sup>+</sup>][BF<sub>4</sub><sup>-</sup>]. For other frequent anions such as [dca<sup>-</sup>], [PF<sub>6</sub><sup>-</sup>], and [NTf<sub>2</sub><sup>-</sup>], a wide variety of toxicity values was found, depending on the paired cation. For instance [dca<sup>-</sup>] combined with [Bmim<sup>+</sup>], [Emim<sup>+</sup>], [Hmim<sup>+</sup>], [MOMmim<sup>+</sup>], [MOEmim<sup>+</sup>], [Omim<sup>+</sup>], [BMPy<sup>+</sup>], [BMPyr<sup>+</sup>], or [P<sub>6,6,6,14</sub><sup>+</sup>] yielded growth rates from 0 to 155%. This fact could be attributed to the dependency of the toxicity values on the IL cation and the possible synergy toxicity effects between the cation and anion.

## Choosing the Less Toxic Ionic Liquids for *Shewanella* sp. Applications

Two general possibilities for the joint application of ILs and *Shewanella* sp. in chemical processes are 1) the use of ILs as reaction media in biotechnological processes or 2) the use of ILs as extractants in biphasic systems with *Shewanella* sp. in an aqueous phase. For the first scenario, the key aspect is to find an IL compatible with this bacterium. The results obtained in this study in terms of biocompatibility have been obtained from a wide range of compounds, but they can also help us to qualitatively predict the toxicity of other nonanalyzed ILs. From this work, we can know that several ILs could serve as a medium for *Shewanella* sp. or could be used in different applications such as microbial fuel cells, in which *Shewanella* sp. act as biocatalysts and ILs are used for membrane preparation. Between the biocompatible ILs, we have found the following: [Hmim<sup>+</sup>][Cl<sup>-</sup>], [MOMmim<sup>+</sup>][BF<sub>4</sub><sup>-</sup>], [MOMmim<sup>+</sup>][dca<sup>-</sup>], [BMPy<sup>+</sup>][BF<sub>4</sub><sup>-</sup>], and [EPy<sup>+</sup>][ETSO<sub>4</sub><sup>-</sup>]. These selected ILs have a growth rate higher than 150% and an inhibition radius equal to zero. It is important to remark that in all cases these ILs were water-soluble, which relates the idea of water solubility to biocompatibility. This is highlighted by the fact that 19 out of 29 insoluble ILs allowed no growth, while only 17 out of 40 water-soluble ILs showed no growth for *Shewanella* sp. Furthermore, only two water-insoluble ILs provided growth rates over 100%, [N<sub>8,8,8,1</sub><sup>+</sup>][CF<sub>3</sub>SO<sub>3</sub><sup>-</sup>] and [P<sub>6,6,6,14</sub><sup>+</sup>][BF<sub>4</sub><sup>-</sup>], and in both cases, the inhibition radius was higher than 1.4 cm.

It is important to note that it is hard to find conventional organic solvents that extract polar compounds from the water with high efficiency since they would need to be water-insoluble and polar, two properties barely possible in conventional organic solvents. These limitations can be overcome in the case of using ILs as extractant medium from water solutions. Furthermore, the ILs would have to be biocompatible with *Shewanella* sp. In this case, the two ILs, [N<sub>8,8,8,1</sub><sup>+</sup>][CF<sub>3</sub>SO<sub>3</sub><sup>-</sup>] and [P<sub>6,6,6,14</sub><sup>+</sup>][BF<sub>4</sub><sup>-</sup>], could meet both conditions.

## Mechanisms of Ionic Liquid Toxicity

Relatively, few mechanisms have been suggested to explain the toxicity of ILs toward marine bacteria, but membrane disruption is considered the most common. ILs at concentrations several orders of magnitude higher than those used in our study have been shown to disrupt synthetic membranes (Sena et al., 2010). In aquatic systems, the side-chain effect could be observed even with the shorter side chains, e.g., IM12 (1-ethyl-3-methyl-imidazolium) and IM14 (1-butyl-3-methyl-imidazolium), which means that the EC50 values for IM12 are higher than
for IM14 (Stolte et al., 2007a; Stolte et al., 2007b). Our results support the membrane disruption mechanism by toxic ILs since, in general, increasing toxicity is observed with increasing IL hydrophobicity and alkyl chain length in the cation (which also contributes to IL hydrophobicity). Furthermore, the inclusion of a heteroatom such as oxygen leads to reducing hydrophobicity and thus toxicity. Nevertheless, other molecular mechanisms cannot be excluded when explaining the toxicity of ILs.

## CONCLUSIONS

This work assesses the toxicity of a high number of ILs toward *Shewanella* sp. in order to analyze their biocompatibility and possible joint application for bioprocess implementation. We observed that the adequate combination of cations and anions can make the whole molecule safe for alive bacterial cells in bioprocess devices. Different and wide ion combinations are possible, by selecting cation-anion combinations suitable to specific applications, which highlights the necessity of screening IL toxicity toward the design of biocompatible compounds.

The results obtained allowed establishing several toxicity-structure relationships. According to the results obtained, the immiscibility of ILs in water has an important effect on the toxicity behavior of the ILs since 66% of the water-immiscible ILs tested were highly toxic, whereas only 42% of immiscible ILs were highly toxic. Furthermore, it was observed that the length of the alkyl chain of the cation presented an important impact on IL toxicity. As a general trend, an increase in the alkyl chain length, which involves an increase in the hydrophobicity, contributes to rising IL toxicity.

It was also determined that several ILs could be used as reaction media in the presence of *Shewanella* sp. based on their biocompatibility and water miscibility, specifically [Hmim⁺][Cl⁻], [MOMmim⁺][BF₄⁻], [MOMmim⁺][dca⁻], [BMPy⁺][BF₄⁻], and [EPy⁺][ETSO₄⁻]. In the case of water-insoluble ILs, [N₈,₈,₈,₁⁺][CF₃SO₃⁻] and [P₆,₆,₆,₁₄⁺][BF₄⁻] showed to be suitable ILs for their use as extractant medium

for *Shewanella* sp. This work demonstrates the potential combination of IL technology and *Shewanella* sp. The results of this study are encouraging and also suggest that new fields could be explored with *Shewanella* sp. and IL materials.

## DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding authors.

## AUTHOR CONTRIBUTIONS

HK: Data curation, formal analysis, investigation, methodology, writing–original draft. AR: conceptualization, resources, funding acquisition, supervision, writing–review and editing. MS and VO: data curation, formal analysis, writing–original draft, writing–review and editing. MK: funding acquisition, supervision. JH: writing–review and editing. FH: conceptualization, funding acquisition, project administration, resources, supervision, writing–original draft, writing–review and editing.

## FUNDING

This work has been funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (Grant number: RTI2018-099011-B-100) and Séneca Foundation (Grant number: 20957/PI/18).

## ACKNOWLEDGMENTS

H. Kebaili thanks Erasmus Mundus Program for the fellowship for students’ exchange received.

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**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Kebaili, Pérez de los Ríos, Salar, Ortiz Martínez, Kameche, Hernández and Hernández Ferández. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Carbon-Based Materials

<figure><img src="image_35.png" /><figcaption>Qiuhong Zhang</figcaption></figure>

Dr. Qiuhong Zhang is a senior scientist at the University of Dayton Research Institute. She has more than 30 years of R&D experience in the fields of nanomaterials, high-temperature polymers, and advanced composite materials. Her main research areas include CNT synthesis, characterization, and application; modification, processing, and manufacturing of advanced nanocomposites based on CNTs; and design, processing, and failure analysis of high-temperature polymer composites. Currently, Dr. Zhang's research focuses on combining high-energy-density carbon-based cathode active materials with high-conductivity carbon nanostructures for high-energy stable lithium-based batteries. Ultimately, efforts are directed toward the development of prototype 3D batteries and scalable technologies.

<caption>Fei Lu</caption>

Dr. Fei Lu graduated from the Department of Optics, Shandong University, in 1985, receiving a doctorate in the science of condensed matter physics in 1997. From 2000 to 2002, as a Hongbao scholar, he worked on rare-earth ion-doped semiconductor light-emitting devices at the Juelich Research Center in Germany. He presided over and undertook the National Natural Science Foundation of China: "Research on New Methods of Ion Implantation in Optical Waveguides", the Ministry of Education's Doctoral Program Fund "Ion Implantation on the Modulation of Ion Exchange KTP Periodic Waveguides", and a number of key projects from the Open Laboratory of Peking University. As the main finisher, he successively participated in one of the key projects of the National Natural Science Foundation of China, three projects of the Natural Science Foundation of China, and one of the doctoral programs of the Ministry of Education. As the main performer, he has won the second prize of National Natural Science, the first prize of Natural Science of Chinese Universities of the Ministry of Education, and the second prize of the Shandong Science and Technology Progress Award. He has undertaken and participated in the declaration of two national invention patents. He has published more than 100 articles.

<figure><img src="image_36.png" /><figcaption>Juana Maria Rosas Martínez</figcaption></figure>
Dr. Juana María Rosas Martínez, a Chemical Engineer at Málaga University since 2003, got her Ph.D. at the same university in 2009. From 2020 to 2015, she has had two postdoctoral contracts at the Complutense University of Madrid and the University of Alicante. After this date, she has worked as Associate Professor at Málaga University. Her main research areas are lignocellulosic waste valorization through thermochemical processes and its application in adsorption and catalytic processes with the aim to reduce or eliminate pollutants in flue gas and to get biofuels and industrial chemicals within a biorefinery context. Besides, she has analyzed the use of electrospray/electrospinning in inorganic catalysts preparation for catalytic applications.

<figure><img src="image_37.png" /><figcaption>Atsuko Sekiguchi</figcaption></figure>

Dr. Atsuko Sekiguchi has been a Senior Researcher at the National Institute of Advanced Industrial Science and Technology since 2015 and is currently a member of the CNT-Application Research Center. She has been a Review Editor for Frontiers in Materials' Carbon-based Materials section since 2016.
<figure><img src="image_38.png" /></figure>

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# Thermal Properties Enhancement of Vertically Aligned Carbon Nanotubes-Based Metal Nanocomposites as Thermal Interface Materials

Qiuhong Zhang<fnref n="1" />*, Brian A. Calderon<fnref n="1" />, Charles R. Ebbing<fnref n="1" />, Levi J. Elston², Larry W. Byrd² and Bang-Hung Tsao²

Increasing power densities in high-power electronic packages require advanced heat transmission from their respective thermal interface materials (TIMs). Modern TIMs do not accommodate both thermal performance and mechanical compliance with increasing device power density. Vertically aligned carbon nanotubes (VACNT) are advantageous in that their intrinsic properties promote both high thermal conductivity while maintaining a mechanically flexible/compliant interface. Therefore, it is a promising approach to use carbon nanotubes (CNTs), specialty VACNTs, to make a novel interface heat transfer material. However, the high thermal contact resistance between VACNTs and substrate(s) has been a big issue to limit its application. In this study, the effect of post-processing techniques such as plasma treatment and surface metallization of VACNT layer (directly grown on Cu substrate) on interfacial properties of CNTs with matching substrate were explored. Thermal properties were evaluated via a Laser Flash testing system. Thermal test results demonstrated that modifying the surface of the VACNT layer is effective method to improve interfacial attachment between CNTs and matching substrate. Results indicated that, among different VACNT surface modification methods, the plasma treated VACNT layer surface promote the best thermal properties of VACNT-based metal nanocomposite as a TIM. Compared to the unmodified VACNT layer, in this study, the interphase thermal resistance of the Cu/VACNT/matched substrate sample made from the plasma treated VACNT layer was reduced approximately 80%.

**Keywords:** vertical aligned carbon nanotubes, plasma, sputtering, metallization, thermal resistance, metal nanocomposite, laser flash testing

## INTRODUCTION

Long-term electronic device failures are often due to thermally induced stress/fatigue that shows up as interfacial delamination. This problem increases the component thermal resistance, which causes increased operational temperature. After enough cycles, this problem propagates into severe delamination that eventually causes the device to overheat, reducing its breakdown voltage within the limits of operation, causing catastrophic failure. Improved thermal management

**OPEN ACCESS**

**Edited by:**

Ruixin Zhu,
Tongji University, China

**Reviewed by:**

Che Azurahanim Che Abdullah,
Putra Malaysia University, Malaysia
Imad Arfaoui,
Université Pierre et Marie Curie,
France

*Correspondence:

Qiuhong Zhang
qiuhong.zhang.3.ctr@us.af.mil

**Specialty section:**

This article was submitted to
Carbon-Based Materials,
a section of the journal
Frontiers in Materials

Received: 15 June 2020

Accepted: 28 September 2020

Published: 28 October 2020

**Citation:**

Zhang Q, A. Calderon B, R. Ebbing C, J. Elston L, W. Byrd L and Tsao B-H (2020) *Thermal Properties Enhancement of Vertically Aligned Carbon Nanotubes-Based Metal Nanocomposites as Thermal Interface Materials*. *Front. Mater.* 7:572956.

<footnote n="1">¹University of Dayton Research Institute (UDRI), Dayton, OH, United States, ²Air Force Research Laboratory (AFRL), WPAFB, Dayton, OH, United States</footnote>
materials, namely thermal interface materials (TIMs) that can maintain a low thermal resistance but extend the device fatigue life, will increase component reliability and reduce operational failures. Thus, TIMs play a key role in thermal management of power modules in high-performance electronic packages. Some major issues with current TIMs such as fatigue failure (solder), low thermal conductivity (phase change materials), and dry out/ pump out (grease) have lead researchers to develop advanced nano-engineered materials to make more efficient thermal interfaces in high power density device. Among the many nano-materials available, carbon nanotubes (CNTs), with their unique thermal, electrical, and mechanical properties, are a very attractive candidate for advanced TIMs (Berber et al., 2000; Che et al., 2000; Kim et al., 2001), and have been widely investigated (Ganguli et al., 2012; Ganguli et al., 2013). It has been reported that taking full advantage of the positive influence of CNTs on the effective thermal conductivity of nanocomposites is a challenge due to difficulties in homogeneous distribution of nanotubes in the matrix phase and proper orientation/alignment for an optimum thermal path (Chu et al., 2010; Firkowska et al., 2011). Therefore, to overcome this problem, significant attention has shifted to using vertically aligned CNT arrays (VACNT) as a promising TIM structure.

An aligned CNT configuration possesses a synergistic combination of mechanical compliance, extraordinarily high axial thermal conductivity (3,000 W/m·K for individual MWCNT), small coefficient of thermal expansion, and high thermal and chemical stability (Berber et al., 2000; Che et al., 2000; Yang et al., 2002; Hu et al., 2006). The elasticity/ conformability feature is particularly advantageous in addressing mismatches in coefficients of thermal expansion that can cause TIM delamination and device failure. In the past several years, VACNT arrays have been widely investigated with successful results from synthesis to characterization (Huang et al., 2005; Xu and Fisher, 2006; Cola et al., 2007; Tong et al., 2007). However, practical implementation of CNTs as TIMs in an electronic package configuration is not a trivial endeavor. A large interfacial thermal resistance has been found between CNTs and the contact substrates, which erects a significant barrier against applying CNTs as TIMs. This resistance is primarily caused by the small percentage of CNTs in contact with the substrate surface and weak interfacial adhesion at each matching surface (Panzer et al., 2008; Son et al., 2008; Lin et al., 2009). Therefore, based on investigation of VACNTs and characterizing the influence on their thermal properties (Wang et al., 2007; Yin et al., 2008; Chen et al., 2010), the fundamental studies on managing the VACNTs/substrate interfaces to effectively reduce the thermal contact resistance and simultaneously improve the interfacial adhesion is necessary and very important. A few groups have explored techniques to enhance the contact area at the CNT-substrate bond line, particularly at the free CNT end (Cola et al., 2008; Cross et al., 2010; Barako et al., 2012). However, the total thermal resistance does not decrease sufficiently due to the material's low intrinsic thermal conductivity (grease), weak bonding (metal), as well as infiltration issues with CNT array. To overcome these problems, the proper

surface modification of VACNTs combined with a high conductivity matrix fill material is a promising approach. Compared to nonaligned CNTs, vertical aligned CNT forest surface modification continues to be a challenge. The difficulties lie in controlling the nanotube length being modified and restricting the nanotube bundling due to the capillary force and van der Waal force between the tubes (entanglements). Among the different methods available to modify/functionalize VACNTs, plasma technique can not only be used to produce a cleaned VACNTs surface (remove amorphous carbon on as-received CNT layer), but can also functionalize CNTs by opening the top end caps or making side wall defects to improve wettability. Using sputter coating techniques, the selected single or multi thin metal layer can be coated on the CNTs surfaces. This suitable thin layer will enhance CNT and metal matrix interfacial properties. On the other hand, compared to pure CNTs, metal coated CNTs with increased mechanical properties and reduced interfacial Van de Waals forces will benefit by maintaining CNT alignment during matrix filtration processing.

Previous work by this group demonstrated that a uniform VACNT layer can be grown directly on a conductive substrate (Cu, Carbon, etc.) when a suitable thin buffer layer was coated on the surface (Zhang et al., 2004). In order to improve the interface connection between the CNTs and the matching substrate, this study focused on the surface modification of the VACNT array grown on the Cu foil substrate (as CNT/Cu), followed by optimum CNT-surface solder filtration processing to form a VACNT-based metal nanocomposite (MNC) structure. This procedure not only increases the VACNT layer to solder/substrate contact, but it also can introduce high thermal conductivity material on the CNTs surface as a bridge to enhance adhesion of VACNTs to a matching substrate surface (ceramic). Furthermore, given the excellent thermal properties of VACNTs in combination with the high electrical and thermal conductivities of metals, VACNT-based MNCs can meet the increasing demands for tailorable performance thermal management materials for use in heat sinks and advanced electronic packages in the aerospace, automotive, and electronics markets.

# MATERIALS AND METHODS

## Sample Preparation

### Synthesis of Vertically Aligned Carbon Nanotubes
Layer on Cu Foil Substrate

The VACNT layer were synthesized directly on a 10 mm × 10 mm × 2.35 mm copper (Cu) substrate (purchased from Online metals) via a FCCVD (Floating Catalyst CVD) method (Zhang et al., 2004). Before the CNT growth process was initiated, an optimized 10–15 nm aluminum (Al) buffer layer was applied to the pre-cleaned Cu substrate surface using a sputter coating technique. The growth process/conditions of CNTs by FCCVD is the same as previously reported (Zhang et al., 2004). The density and length of VACNT layer on Cu foil was controlled by growth time under the same growth conditions (Zhang et al., 2004). In this study, VACNT/Cu samples, with a VACNT layer thickness between
<figure><img src="image_39.png" /><figcaption>**FIGURE 1** | (A) Top view setup for VACNT layer surface morphology. (B) Side view setup for VACNT layer thickness.</figcaption></figure>

80 and 100 µm, were selected to prepare a series of three-layer (sandwich) samples. SEM (JEOL JSM6060 Scanning electron microscope) was employed to characterize the morphology/structure of VACNT layer. An example setup is shown in **Figure 1**.

## Vertically Aligned Carbon Nanotubes Layer Surface Modification

Owing to its intrinsic structure and the high packing density of the VACNT layer, the main issue faced in the fabrication of VACNT-based MNC is the low wettability of CNTs with the molten metal matrix fill. To overcome this problem, in this study, the surface modification techniques were introduced for the objective of enhancing overall attachment between the VACNT layer and matching substrate. The different methods including plasma (South bay Technologies RIE-2000), sputter coating (Denton DV502A), and a combination of plasma and sputter coating were selected to modify the VACNT layer surface to improve CNT surface wettability while maintaining the alignment of VACNTs. The time of plasma treatment and the thickness of the metal layer were varied to find the optimum conditions for surface modification of the VACNT layer. Before

and after surface modification, the VACNT layer surface morphology/structure was characterized by SEM. The results will be discussed in the next section.

## Fabrication of Vertically Aligned Carbon Nanotubes-Based Metal Nanocomposite as Thermal Interface Material

Based on VACNT/Cu sample, a three-layer “sandwich” structure (Cu/TIM/Substrate) was created to make the VACNT-based MNC as TIM and processing scheme can be seen in **Figure 2**. The material and size/thickness of each layer on sandwich sample was shown in **Table 1**.

To reach the aforementioned goal, a new soldering setup was developed by converting a Thermo-Electric Generator (TEG) test stand into a Heated Compression System shown in **Figure 3**. A sandwich sample, produced by attaching a thin ceramic (0.64 mm) substrate (coated with a 20 nm nickel and 50 nm gold bi-layer) to a VACNT/Cu sample using selected 241 solder (10 × 10 mm), was first placed into a custom Macor sample holder (see **Figure 3**) and then delivered the holder to the HCS. A developed optimum processing, as **Table 2** shown, was used to fabricate the

<figure><img src="image_40.png" /><figcaption>**FIGURE 2** | Sandwich sample fabrication process and configuration.</figcaption></figure>
<table><caption>**TABLE 1** | Sample preparation materials.</caption><thead><tr><th>Layer</th><th>Material</th><th>Size/Thickness (mm)</th><th>Surface modification/Treatment</th></tr></thead><tbody><tr><td>Target substrate</td><td>Al<sub>2</sub>O<sub>3</sub> Ceramic (Stellar Ceramics)</td><td>10 × 10 × 0.64</td><td>Coated with Ni (20 nm) and Au (50 nm)</td></tr><tr><td>Interphase layer (MNC)</td><td>Solder #241 (95.5 Sn/3.5 Ag/0.7 Cu) (Indium Corp)</td><td>10 × 10 × 0.05 (as received)</td><td>n/a</td></tr><tr><td></td><td>VACNT layer</td><td>10 × 10 × 0.08–0.10 (as received)</td><td>None/plasma/Ni/Ti metallization</td></tr><tr><td>Growth substrate</td><td>Cu</td><td>10 × 10 × 2.35</td><td>As-received, coated with Al buffer layer (15 nm)</td></tr></tbody></table>

<figure><img src="image_41.png" /><figcaption>**FIGURE 3** | Heated Compression System (HCS) for "sandwich" structure MNC fabrication and sample holder with sample in-place (Right bottom).</figcaption></figure>

VACNT based MNC interphase in a three-layer "sandwich" sample. The desired temperatures and load pressures are monitored/controlled using a custom LabVIEW program; a nitrogen purge capability was added to ensure the copper does not oxidize at high temperatures. Using metal coated ceramic substrates, solder241 and VACNT/Cu samples (with/without VACNT layer surface modification), a series of sandwich samples were produced by HCS (Figure 3) under the optimized processing conditions (Table 2). The structure of sandwich and morphology of interphase (MNC) in sandwich sample was characterized by SEM and will be discussed in next section.

## Vertically Aligned Carbon Nanotubes/ Solder Interphase (Metal Nanocomposite) Thermal Analysis and Stability Testing

The thermal diffusivity of the VACNT/solder nanocomposite interphase layer was conducted by utilizing the Netzch Laser

Flash Apparatus 457 (LFA) with the goal of characterizing thermal properties and evaluating thermal performance/ stability. In this study, a series of three-layer sandwich samples, made with different treatments on the surface of the VACNT layer (plasma, sputtering, etc.), were tested. Each sample was tested at least three times in alternating tray positions with a one-dimensional heat flux at temperature steps of 25, 50, 100, and 150°C. For three-layer structure samples, the Cu (bottom) and ceramic (top) substrates are programmed as known layers with a constant thickness and density; as the unknown layer, the thickness of VACNT based MNC was measured with SEM. The standard heat-loss (minus contact resistance) correction model was selected as the best match to determine the correct diffusivity (Firkowska et al., 2011).

The reliability of the VACNT based MNC were evaluated by a custom designed Rapid Thermal Cycling System. All three-layer (Cu/MNC/Ceramic) samples which were already measured in the LFA, were tested. To simulate the conditions that TIMs undergo in the field, the experimental conditions were selected, seen in Table 3. Thermal properties of tested samples were measured/ calculated by LFA system again after each set of 50 thermal cycles from -25 to 125°C up to 150 cycles. The thermal stability of interphase was assessed by a percentage increase in the thermal resistance of MNC after thermal cycles. According to a standard approach to testing device failure in electronics, a 50% increase in

<table><caption>**TABLE 2** | Optimized parameters for VACNT based MNC fabrication (HCS).</caption><tr><td>Pressure (load force)</td><td>0.9 MPa (20–40 lbs)</td></tr><tr><td>Temperature</td><td>~20°C above solder melting point</td></tr><tr><td>Duration of hold</td><td>3–5 min</td></tr><tr><td>Sample set-up</td><td>Hot side: Cu/Cold side: ceramic</td></tr></table>
<table><caption>**TABLE 3** | Parameters of thermal reliability testing (rapid thermal cycling machine).</caption><tr><td>Temperature range</td><td>-25 to 125°C</td></tr><tr><td>Temperature ramp rate</td><td>3.3°C/min (low ramp rate)</td></tr><tr><td>Soak time (per set point)</td><td>3 min</td></tr><tr><td>Number of cycles per run</td><td>50 cycles</td></tr></table>

the thermal resistance of the MNC indicates failure of the sample because this can lead to further degradation of the interface and thermal runaway as the mismatch in thermal expansion becomes worse.

# RESULTS AND DISCUSSION

## The Morphology of Vertically Aligned Carbon Nanotubes Layer Before and After Surface Modification

As a TIM, the CNT layer structure plays an important role on its thermal and mechanical properties. The VACNT layer with high packing density will increase interface contact area and resulted a good thermal and mechanical performance. SEM images of CNT array synthesized on Cu substrate are shown in **Figure 4**. The result indicated that, using FCCVD method, a uniform and high dense vertical aligned CNT layer was successfully grown on buffer layer coated Cu foil surface. After plasma or/and sputtering metallization treatment, the surface of VACNT layer has undergone significant changes.

## Vertically Aligned Carbon Nanotubes Layer Surface Plasma Etching

Several CNT/Cu samples were subjected to plasma treatment (H₂ as sources) with 5, 10, or 15 min of treating times. The changes on VACNT layer surface morphology before and after treatment as well as the effect of treated time were monitored with SEM, and the results are shown in **Figures 5 and 6**. Comparing the SEM images before and after plasma etching, in **Figure 5**, the surface morphology of the VACNT layer obvious changed as expected; when the treatment duration was increased from 5 to 15 min, more “open space” at the top became exposed. There was a visible difference between the 10 and 15 min plasma treatment (see **Figure 6**): the 10 min

treatment tops off between 1 and 2 microns from the surface whereas the 15 min treatment shreds through 2–3 microns from the surface, leaving a much lower density of VACNT layer packing and thus further increasing the solder infiltration surface. This result indicated that the surface morphology of the treated VACNT layer was determined by the time of plasma treatment. There was more open space on the top of the VACNT layer after a long-time processing, which will result in the improved wettability of the CNT array.

## Vertically Aligned Carbon Nanotubes Layer Surface Metallization

The metallization of both substrate and VACNT layer surface is a promising approach to reduce the thermal contact resistance and simultaneously improve the interfacial adhesion of CNTs/matching substrate. In this study, Ti (Titanium)) and Ni (Nickel) were selected to metalize/ modify the VACNT layer surface of VACNT/Cu samples due to their excellent physical properties and good adhesion with CNT (Ti as first layer) and solder (Ni as second layer). The Ti/Ni bi-metal thin layer with different thickness and Ti/ Ni ratio were sputter coated on VACNT layer surface of VACNT/Cu samples. SEM images of the VACNT layer surface, before and after metal coating, can be found in **Figure 7**. These images show that a thin metal layer was evenly covered on the surface of the VACNT layer, which will result in a strong interfacial adhesion between the CNT array and the solder. The sputtering method was also used to coat a Ni/Au (20 nm/50 nm) double-layer thin film onto the surface of ceramic matching substrate to enhance interface properties between VACNT layer and substrate.

## Vertically Aligned Carbon Nanotubes Layer Surface Plasma Treatment and Metallization

Surface metallization and plasma treatment are the two methods of surface modification pursued in this study. Ideally, plasma treatment will promote wettability/solder infiltration and surface metallization will improve surface attachment. Therefore, if these two methods are combined for VACNT layer surface treatment, the best interface performance should be produced. Based on this consideration, the sample processing was performed by coating different thicknesses of Ti/ Ni bi-metal layer on the plasma-treated (via H₂ for 10 or

<figure><img src="image_42.png" /><figcaption>**FIGURE 4** | SEM images of VACNTs grown on 15 nm Al coated Cu foil substrate. Top view (Left, scale bar = 5 µm) and side view (Right, scale bar = 50 µm).</figcaption></figure>
<figure><img src="image_43.png" /><figcaption>**FIGURE 5** | Top view SEM images of VACNT layer: before and after 15 min plasma treatment. (Scale bar from left to right = 500, 10, 1.0, 0.5 µm.)</figcaption></figure>

15 min) VACNT layer surface. The SEM images of the combined surface treatment samples can be found in **Figure 8**. The SEM images depict the expected results: open areas on the surface of VACT layer for solder infiltration are covered with a layer of metallic particles.

## Structure of Three-Layer “Sandwich” Sample with Vertically Aligned Carbon Nanotubes-Based Metal Nanocomposite

The structure of sandwich and morphology of interphase (MNC) in sandwich sample was characterized by SEM and shown in **Figure 9**. SEM images shown a good interface connection between the MNC and the two substrates (ceramic and Cu). The results demonstrated that, with a suitable solder material, the

fabrication process of sandwich sample can not only completely attach the matching substrate to the surface of the VACNT layer, but also maintain the interface adhesion of VACNT/Cu (No CNTs peeled from Cu foil). Due to the elastic properties of the VACNT array, the deformation of the VACNT layer can be clearly observed in the SEM image of **Figure 9**. As a TIM, this deformation should bring MNC good thermal and mechanical performance, especially in terms of thermal reliability.

## Thermal Property of Vertically Aligned Carbon Nanotubes-Based Metal Nanocomposite in Sandwich Sample

Thermal property analysis for the MNC interphase of Cu/MNC/Ceramic samples focused on LFA testing to calculate the thermal

<figure><img src="image_44.png" /><figcaption>**FIGURE 6** | Top view SEM images of VACNT layer: comparison of plasma treatment durations. (Scale bar on top = 10 µm; Scale bar on bottom = 1.0 µm.)</figcaption></figure>
<figure><img src="image_45.png" /><figcaption>**FIGURE 7** | Top view of SEM images: the metalized VACNTs surface by Ti/Ni bi-metal layer. (Scale bar = 1.0 µm)</figcaption></figure>

<figure><img src="image_46.png" /><figcaption>**FIGURE 8** | Top view SEM Images. (A) The bi-metal layer with different thickness coated on Plasma treated VACNTs surface (10 min). (B) The bi-metal layer coated on Plasma treated (10 and 15 min) VACNTs surface. Scale bar = 1 µm (A); Scale bar (from left to right) = 1, 50, 1.0, 0.5 µm (B).</figcaption></figure>
<figure><img src="image_47.png" /><figcaption>FIGURE 9 | Side view of fabricated sandwich sample (top) and MNC interphase (bottom). (Top scale bar = 500 µm; Bottom scale bar = 50 µm).</figcaption></figure>

resistance of the VACNT/solder interphase layer. All qualified three-layer sandwich samples, including with and without VACNT layer surface modification, were tested. Thus, the following results and discussion section focused on how the thermal properties of the MNC interphase were influenced by the method of VACNT layer surface treatment. Specifically, the thermal resistance was calculated based on the thermal diffusivity measured at 100°C by Eq. 1, where L is the VACNT layer thickness, α is the measured diffusivity, ρ is the VACNT layer density and C$_{p}$ is the specific heat of the CNTs set to a constant of C$_{p}$ = 0.8.

$$R = \frac{L}{\alpha \rho C_p} \qquad (1)$$

To minimize error, numerous samples (grouped by VACNT layer surface treatment method) were tested. The average thermal resistance of each group samples, with standard deviation, can be found in Figure 10. These results will be discussed based on the VACNT layer surface treatment method.

From test results in Figure 10, the following conclusions about the effect of VACNT layer surface modification on the thermal resistance of the interphase (Cu/VACNT-solder/Ceramic sample) were drawn:

(1) The thermal resistance of MNC interphase was clearly influenced by surface treatment of VACNT layer. The changes of interface contact area, infiltration space and CNT surface wettability after surface treatment are the main reasons for this result.

(2) The thermal resistance of all samples made of untreated VACNT layers varied greatly (a large standard deviation). The main reason is most likely due to the uncertainty of the surface structure (morphology/purity, etc.) with untreated VACNT layer (as received). These variations will cause actual the interface contact area between the VACNT layer and matching substrate to be different varying for each untreated sample, thereby resulting in different thermal resistance values.

Compared to surface modified VACNT layer, the sample made with surface unmodified VACNT Layer had the highest thermal resistance value (see Figure 10). Namely, without

VACNT layer surface modification, there is a large interface resistance between VACNT layer and matching substrate although fabrication techniques were optimized. This issue is primarily caused by the lack of solder infiltration within the VACNT layer during fabrication processing due to the CNTs low wettability and dense surface (non-aligned) of VACNT layer.

Surface metallization promotes improved adhesion (wettability) for contact between the CNT array tips and solder. Improved interfacial adhesion, where the thickness of metallization layer plays an important role, establishes a better connection, and thus resulted in improved thermal and mechanical properties. In this study, the interfacial mechanical properties (including the tensile strength and shear strength) of sandwich samples were explored by pull-off (tensile) and shear test. The results indicated that, when an 800 nm bi-metal thin layer (200 nm Ti layer and then 600 nm Ni layer) was coated on VACNT layer surface, a strong tensile strength was produced in the interface between VACNT layer and matching substrate. Therefrom, the 800 nm bi-metal layer was applied for all VACNT layer surfaces metallization. It can be clearly seen in Figure 10 that MNC interphase thermal resistance was reduced by VACNT surface metallization. Adding conducting film/particles to the VACNT layer surface to enhance adhesion between CNT-solder are main reason for this improvement. However, an average thermal resistance of 66 mm²·K/W means that surface metallization does not significantly improve the interphase thermal resistance. This is most likely due to the small penetration space for solder on the surface of the metalized VACNT layer.

VACNT layer surface plasma treatment was applied to improve the overall wettability of the CNT surface and opened space to enhance solder infiltration during MNC fabrication. Compared to the reference of VACNT based MNC (untreated) thermal resistance, the results as shown in Figure 10 demonstrated a significantly lower thermal resistance with surface plasma modified VACNT/solder interphase (between two substrates). Specifically, samples which were treated by 15 min H₂ plasma had the lowest thermal resistances with a small standard deviation, as well as a greater improvement compared to sample made of 10 min H₂ plasma treated
<figure><img src="image_48.png" /><figcaption>**FIGURE 10** | Thermal resistance of MNC interphase samples, per category of surface modification.</figcaption></figure>

VACNT layer. The reasonable explanation for this result is that the longer treatment time can create more space on VACNT layer surface as shown in **Figure 6**, and this would allow more solder to infiltrate into the VACNT array during three layer sample fabrication to produce a stable MNC interface.

VACNT layer surface plasma treatment followed by surface metallization processing were expected to enhance the interfacial adhesion of CNT tip and solder while providing more infiltration space to improve thermal properties and reliability of the VACNT based MNC as TIM. The experimental results in **Figure 10** indicated that the surface modification of the VACNT layer by a combination technique (plasma and metallization) did reduce the thermal resistance of the VACNT based MNC interphase compared to the original (untreated samples). The pull-off testing results also show a strong interfacial adhesion between the modified VACNT surfaces and matching substrate. Under the same metallization

conditions (metal layer Ti/Ni = 200 nm/600 nm), the VACNT surface after 15 min of H₂ plasma treatment produced a lower MNC interphase thermal resistance than that with 10 min of H₂ plasma treatment. However, this value is much higher than that of samples made with VACNT layer treated by plasma only. This is because the metal layer/particles coated on plasma treated VACNT surface (see **Figure 8**) may prevent solder infiltrating into the VACNT layer during MNC fabrication processing.

## Thermal Performance/Reliability of Vertically Aligned Carbon Nanotubes-Based Metal Nanocomposite in Sandwich Sample

Although reducing the overall thermal resistance is a primary goal of this study, practical applications will cause periodic thermal stresses during normal operations. Thus, reliability

<figure><img src="image_49.png" /><figcaption>**FIGURE 11** | Thermal cycles vs. thermal resistance of VACNT-based MNC as thermal interphase.</figcaption></figure>
<table><caption>TABLE 4 | The effect of thermal cycles on thermal resistance (average) of TIM.</caption><thead><tr><th>interphase<br/>/Resistance<br/>mm 2·K/W</th><th>Solder only<br/>(no VACNT layer)</th><th>Untreated VACNT layer</th><th>Metallized VACNT layer<br/>(800 nm)</th><th>Plasma (P)(10 min)<br/>VACNT layer</th><th>Plasma(P)(15 min)<br/>VACNT layer</th><th>P/M (10 min/800 nm)<br/>VACNT layer</th><th>P/M (15 min/800 nm)<br/>VACNT layer</th></tr></thead><tbody><tr><td>R₀ (0 cycles)</td><td>4.95</td><td>77.81</td><td>66.35</td><td>12.45</td><td>5.90</td><td>47.70</td><td>36.40</td></tr><tr><td>R50 (50 cycles)</td><td>11.05</td><td>88.95</td><td>87.3</td><td>14.15</td><td>6.65</td><td>52.40</td><td>41.30</td></tr><tr><td>ΔR(R - R₀)</td><td>6.1</td><td>11.14</td><td>20.95</td><td>1.7</td><td>0.75</td><td>4.7</td><td>4.9</td></tr><tr><td>ΔR/R₀% increase</td><td>123%</td><td>14.3%</td><td>31.57%</td><td>13.65%</td><td>12.71%</td><td>9.90%</td><td>13.46%</td></tr><tr><td>R100 (100 cycles)</td><td>—</td><td>113.62</td><td>77.75</td><td>15.56</td><td>7.14</td><td>53.80</td><td>43.67</td></tr><tr><td>ΔR(R - R₀)</td><td>—</td><td>35.81</td><td>11.4</td><td>3.11</td><td>1.24</td><td>6.10</td><td>7.27</td></tr><tr><td>ΔR/R₀% increase</td><td>—</td><td>46.00%</td><td>47.32%</td><td>24.98%</td><td>20.93%</td><td>12.79%</td><td>14.48%</td></tr><tr><td>R150 (150 cycles)</td><td>—</td><td>132.20</td><td>101.30</td><td>17.60</td><td>8.28</td><td>53.8</td><td>49.4</td></tr><tr><td>ΔR(R - R₀)</td><td>—</td><td>54.39</td><td>34.95</td><td>5.15</td><td>2.38</td><td>6.10</td><td>13.00</td></tr><tr><td>ΔR/R₀% increase</td><td>—</td><td>66.90%</td><td>52.67%</td><td>41.37%</td><td>40.25%</td><td>12.79%</td><td>35.71%</td></tr></tbody></table>

Bold entries show the increased percentage of MNC interphase thermal resistance after thermal cycling and italic entries show the increased thermal resistance of MNC interphase after thermal cycling.

testing is necessary to characterize the thermal and mechanical performance of three-layer samples being created with VACNTs based nanocomposite (as TIM). In this study, the reliability of the VACNT-based MNC was evaluated as the percent increase in VACNTs/solder interphase thermal resistance from the original value through nominally induced thermal stress (thermal cycling). Using the aforementioned procedure and equipment related to reliability testing, all three-layer samples were subjected to 150 thermal cycles from -25 to 125°C. The thermal performance of the samples was retested by LFA after each set of 50 thermal cycles. The thermal resistance (including the results before and after the thermal cycle) and the nominal percentage increase (original value as reference) in thermal resistance after each set of 50 thermal cycles were illustrated in Figure 11 and Table 4, respectively, by surface treatment category as below.

To compare the thermal properties of VACNT-based MNC and conventional TIM (solder), the Cu/Solder/Ceramic three-layer sandwich samples were prepared and tested. As expected, the solder-only TIM produced the lowest value of thermal resistance (good thermal conductivity) but quickly loses its reliability (very poor interfacial tensile strength) after only 50 thermal cycles, and the thermal resistance was significantly increased by more than 100% as shown in Figure 11 and Table 4. As compared, the sandwich samples made of unmodified VACNT layer produced a more reliable VACNT-based TIM, which showed the lower rate of increase in thermal resistance after thermal cycles as seen in Figure 11 and Table 4. However, the overall performance of these samples was not satisfactory due to their high thermal resistance.

Related to reliability during thermal cycling, all sandwich samples made with surface modified VACNT layer were tested. As expected, thermal resistance of MNC interphase for all tested samples increased after each thermal cycling test. However, the samples which have the VACNT surface modified, except for the samples with surface metallization treatment, showed modest increases in thermal resistance when compared to samples which have not, especially after 100 cycles. Except the surface metallized samples, all samples made with surface modified VACNT layer demonstrated improved interphase thermal stability, resulting in the thermal resistance increased by less than 50% (failure point) even after 150 thermal cycles. Moreover, the samples made with both plasma treated and metalized VACNT layer demonstrated the best thermal stability by showing a 12.79% increase on thermal resistance after 150 thermal cycles. The good thermomechanical performance under continued stresses was attributed to 1) opened veins/space (plasma treatment) on the VACNT array for solder infiltration; 2) metal particles (metallization) to promote the wettability of CNT tips to form a strong interfacial adhesion of VACNT layer and solder.

## CONCLUSION

In this study, a VACNT layer was synthesized directly onto Cu foil substrate by FCCVD method. In order to achieve better interfacial adhesion of VACNT-based MNC (as TIM), a VACNT layer surface post-processing procedure (including plasma treatment and
metallization) was developed to improve the solder infiltration onto VACNT layer and thereby improve the overall thermal performance of interphase. The effect of different post-processing techniques on VACNT layer surface and interfacial properties of VACNTs/matching substrate were explored. The thermal performance of MNC were evaluated via Laser Flash system and Rapid Thermal Cycling System. Overall experimental results demonstrated:

(1) The VACNT layer surface modification is an effective method to improve thermal properties of VACNT based MNC as TIM. Compared with surface untreated VACNT layer, all the surface modified VACNT layer (as TIM) showed lower thermal resistance and higher thermal stability in tested sandwich samples.

(2) Among different VACNT surface modification methods, the plasma treated (especially with 15 min) VACNT layer surface promoted the best thermal properties of MNC, which was manifested by the lowest thermal resistance (5.90 mm²·k/W, close to tested solder value 4.95 mm²·k/W) and high thermal reliability under the thermal cycling test.

(3) Compared with the untreated VACNT layer, the 15 min plasma treated VACNT layer can reduce the thermal resistance of VACNT-based MNC by more than 80%. This finding indicated that CNT surface plasma treatment is a promising technique to enhance interfacial connection between CNT layer and solder to produce a good performance of VACNT based MNC as TIM.

(4) The sample, made with a VACNT layer surface treated by both plasma and metallization, showed an improved thermomechanical performance under thermal cycling.

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## DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

## AUTHOR CONTRIBUTIONS

QZ is a main author, all others are equal. All authors contributed to manuscript revision, read, and approved the submitted version.

## FUNDING

Air Force Office of Scientific Research for funding support.

## ACKNOWLEDGMENTS

The authors would like to thank the Air Force Office of Scientific Research for funding support, the Air Force Research Laboratory/RQQM, and following people for their help on this project: John Murphy, Jacob Lawson, Ammon Williams, James Scofield, William Lanter, and Joseph Merrett.

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