Critical Reviews in Biotechnology

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Microbial surfactants in nanotechnology: recent

trends and applications

Marcia Nitschke & Crisiane Aparecida Marangon

To cite this article: Marcia Nitschke & Crisiane Aparecida Marangon (2021): Microbial surfactants
in nanotechnology: recent trends and applications, Critical Reviews in Biotechnology, DOI:
10.1080/07388551.2021.1933890

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CRITICAL REVIEWS IN BIOTECHNOLOGY
https://doi.org/10.1080/07388551.2021.1933890

REVIEW ARTICLE

Microbial surfactants in nanotechnology: recent trends and applications

Marcia Nitschkea and Crisiane Aparecida Marangonb

a
Departamento F
ısico-Qu
ımica, Instituto de Qu
ımica de S~ao Carlos (IQSC) – USP, S~ao Carlos, Brazil; bPrograma Interunidades
Bioengenharia (EESC/FMRP/IQSC), Universidade de S~ao Paulo (USP), S~ao Carlos, Brazil

ABSTRACT ARTICLE HISTORY

The interest in nano-sized materials to develop novel products has increased exponentially in Received 10 December 2020
the last decade, together with the search for green methods for their synthesis. An alternative to Revised 25 March 2021
contribute to a more sustainable approach is the use of microbial-derived molecules to assist Accepted 9 April 2021
nanomaterial synthesis. In this sense, biosurfactants (BSs) have emerged as eco-friendly substi-
KEYWORDS
tutes in nano-sized materials preparation. The inherent amphiphilic and self-assembly character Biosurfactant; nanoparticle;
of BSs associated with their low eco-toxicity, biodegradability, biocompatibility, structural diver- nanomaterial; nanostruc-
sity, biological activity, and production from renewable resources are potential advantages over ture; nanobiotechnology;
chemically-derived surfactants. In nanotechnology, these versatile molecules play multiple roles. rhamnolipid; surfactin
In nanoparticle (NP) synthesis, they act as capping and reducing agents and they also provide
self-assembly structures to encapsulation, functionalization, or templates and act as emulsifiers in
nanoemulsions. Moreover, BSs can also play as active compounds owing to their intrinsic bio-
logical properties. This review presents the recent trends in the development of BS-based nano-
structures and their biomedical and environmental applications. Fundamental aspects regarding
their antimicrobial and anticancer activities are also discussed.

GRAPHICAL ABSTRACT

Introduction to create new products has increased exponentially in

Nanotechnology is a general term used to describe the
design, production, and application of structures at the
nanoscale level (usually <100 nm but not limited to)
which can be explored in a diverse range of industrial
fields from electronics to health care [1,2]. Manipulation
at the molecular level results in physical, chemical,
magnetic, and structural properties that are not present
on the macroscale; representing an important tool for
the development of new functional nanomaterials [3,4].
Actually, the research and exploitation of nanomaterials
the last years [5]. The synthesis of nanomaterials gener-
ally involves chemical and physical methods that gener-
ate toxic by-products hazardous to both health and the
environment [1,3]. An approach to reduce the possible
risks associated with nanomaterials is the use of green
methods for their synthesis, replacing hazardous chemi-
cals with sustainable and environmental-friendly
reagents [6]. Therefore, the use of bionanotechnology
concepts to obtain nanomaterials using biological
methods emerges as a potential green alternative. The
literature describes several examples of nanomaterial

CONTACT Marcia Nitschke nitschke@iqsc.usp.br Departamento F
ısico-Qu
ımica, Instituto de Qu
ımica de S~ao Carlos (IQSC) – USP, Av. Trabalhador
S~ao Carlense, 400, PO Box 780, Zip code 13560-970, S~ao Carlos, SP, Brazil
*Present address: National Nanotechnology Laboratory for Agribusiness, Embrapa Instrumentaç~ao, XV de Novembro Street, 1452, Zip code 13560–979,
S~ao Carlos, SP, Brazil.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group
2 M. NITSCHKE AND C. A. MARANGON
biosynthesis by microbial cells [7,8] or by using micro-
bial and plant metabolites [9,10]. Strains of bacteria,
fungi, yeasts, and algae can synthesize metallic nano-
particles (NPs) even extracellularly or intracellularly [11]
usually by the enzymatic reduction of metal salts of sil-
ver, gold, platinum, and iron amongst others [7].
Although the use of microbes as nanofactories has sev-
eral advantages, controlling such parameters as size,
shape, and polydispersity [9], combined with the com-
plexity of downstream process [3], are challenges to be
overcome. An alternative to contribute to sustainable
and green methods is to utilize microbial-derived mole-
cules to assist with the nanomaterial synthesis.
Biomolecules that can self-assemble like proteins, pepti-
des, lipids, fatty acids, DNA, or surfactants can act as
templates to nanostructures synthesis [12,13].
Surfactants are required for the preparation of NP in
traditional chemical methods as they reduce aggrega-
tion and increase their stability [2]. However, their syn-
thetic nature does not fit current market needs. In this
view, the use of natural surfactants has emerged as an
eco-friendly alternative to nano-sized material
preparations.
Why biosurfactants?
Microbial surfactants, also known as biosurfactants
(BSs), are surface-active compounds produced by pro-
karyotic and eukaryotic microorganisms, such as bac-
teria [14], molds, and yeasts [15]. Similar to synthetic
surfactants, they contain at least one polar (hydrophilic)
moiety together with an apolar (hydrophobic) group
and such amphiphilic nature allows BS molecules to
align at interfaces of different polarities decreasing sur-
face and interfacial tension [16]. The polar groups pre-
sent on BS usually comprise esters, hydroxyls,
phosphate, carboxyl groups, carbohydrates, peptides,
or proteins, whereas the hydrophobic portion is satu-
rated or unsaturated fatty acids, hydroxy fatty acids, or
fat alcohols [17]. Most BSs are anionic or neutral and
they are classified according to their chemical compos-
ition and molecular mass, as low (e.g. glycolipids and
lipopeptides) and high molecular weight (e.g. lipopoly-
saccharides and lipoproteins) surfactants. Low molecu-
lar weight BS are more effective as surface-active
agents than high molecular mass BS that are good
emulsifiers [18]. Table 1 summarizes the chemical struc-
tures and microbial origin of some BS discussed in
this work.
BS presents low surface and interfacial tension, low
CMC, is able to adsorb at air/water, liquid/liquid, and
solid/liquid interfaces [19]. The physicochemical
properties of BS are not within the scope of this review
however, there are several articles focusing on such
general aspects available in the literature [16,19–21].
Although BSs have similar functions compared to
synthetics, their origin impacts significantly on proper-
ties. BSs are considered environmental friendly mole-
cules owing to their low eco-toxicity and
biodegradability [22]. Additional beneficial properties
including biocompatibility, structural diversity, bio-
logical activity, and production from renewable resour-
ces are potential advantages over their chemical
counterparts [20,23,24]. These versatile molecules can
be applied as emulsifiers, demulsifiers, wetting, and
foaming agents, solubilizers, viscosity reducers, and
cleansing agents in different industrial fields.
Traditionally, the BSs have been utilized in enhanced oil
recovery and bioremediation of oil spills in petroleum
industry and for the bioremediation of metals and other
pollutants [25]. However, their unique features are
attracting attention in cosmetics [26], biomedical [27],
food [23], agriculture [28], and household and laundry
[29] sectors, to name a few. The demand for eco-
friendly and green surfactants increased considerably
during the last years because of the environmental and
health protection awareness of society. BS production
fulfills many principles of green chemistry thus, they
represent an important tool for innovation and sustain-
ability [30].
Production costs are considered an important barrier
to the widespread use of BS. However; the use of inex-
pensive substrates [31], overproducing strains [32],
metabolic engineering techniques [33], effective down-
stream processes [34], and the co-production of BS with
other biomolecules are some strategies that have been
proposed to develop BS production competitively [35].
Actually, efforts to enhance the BS economy are show-
ing results. Rhamnolipids (RLs) production costs, which
have been estimated in the past to be 1000 times as
expensive as the conventional surfactants, are now
10–30 times higher than synthetics thus, they are
becoming suitable for large-scale exploitation [36]. In
addition, refined applications (medical and pharmaceut-
ical) are a potential approach to overshadow their eco-
nomics considering that the benefits of using the BS
can compensate their costs [37]. The microbial surfac-
tants market is valued at 17 million USD in 2020 and is
expected to reach 23 million USD by the end of 2026
[38]. At present, companies such Akzo Nobel, BASF,
Clariant, and Evonik include BS in their portfolio, an
indication of their market trend in the near future. The
use of heterologous expression techniques and enzym-
atic synthesis [39] leads to significant innovations in BS
 CRITICAL REVIEWS IN BIOTECHNOLOGY 3

Table 1. Classification, origin, and chemical structures of some microbial surfactants.

Class Type Origin

Glycolipids Rhamnolipids P. aeruginosa, Pseudomonas sp., Burkholderia sp.
Sophorolipids Candida bombicola, C. apicola, Torulopsis petrophilum
Trehalolipids Rhodococcus erythropolis, Mycobacterium sp.,
Arthrobacter sp.
Mannosyl erythritol Candida antarctica, Pseudozyma sp., Ustilago maydis
lipids (MEL)

Rhamnolipid (mono) Trehalolipid MEL

Sophorolipid
Lipopeptides Surfactin Bacillus subtilis
Iturin A B. subtilis
Fengycin B. subtilis
Lichenysin B. licheniformis

Iturin Fengycin Lichenysin

Surfactin
Polymeric surfactants Liposan Candida lipolytica
Emulsan Acinetobacter calcoaceticus, Arthrobacter calcoaceticus
Lipoproteins Pseudomonas gessardii
Carbohydrate-protein Pseudomonas fluorescens, Debaryomyces polymorphus
lipid

Emulsan

market. The startup company GlycosurfV R (https://www.
glycosurf.com/) offers bioinspired tailor-made glycoli-
pids designed for specific applications. The sugar moi-
ety, carbon chain type, and length can be handled to
attain the desired physicochemical/biological properties
along with high purity, single homolog glycolipids that
are prepared to fulfill client needs. Designing BS with
high purity and single composition can result in more
active/specific compounds that can be applied in low
quantities rendering them even more competitive for
industrial purposes.
The amphiphilic and self-assembly nature of surfac-
tants are very useful properties to obtain nanomaterials.
Although most studies report the use of chemical-
based surfactants, there is an ongoing demand to
replace them by bio-based molecules as described
above. In the next sections, we discuss the recent
developments regarding the use of BS in nanomaterials
synthesis and their applications (Figure 1).
Application of BS in nanotechnology
BS in nanoparticles (NPs) synthesis
Metal nanoparticles
Chemical and physical methods to produce NPs are
widely described and some disadvantages pointed out
4 M. NITSCHKE AND C. A. MARANGON
Figure 1. General overview of BS applications in nanotechnology.
include the generation of toxic chemicals, use of high
temperature and pressure, requirement of costly equip-
ment amongst others [1]. The most utilized methods
for the synthesis of metal NP are chemical reduction
and microemulsion-based methods. In such techniques,
surfactants are incorporated into the formulation as
capping agents to prevent particles from agglomer-
ation even by the electrostatic or steric stabilization of
NP surfaces [40]. Thus, surfactants are key elements to
stabilize NP growth, favoring their uniform dispersion in
solution. The surfactant adsorption into metal NP surfa-
ces is the central event to stabilization and depends on
the type of surfactant employed (ionic, nonionic, and
polymeric) and the thickness of the adsorbed layer
[40,41]. One of the most important issues to green NP
synthesis is the use of non-hazardous reagents as
reducing and stabilizing agents [42]. Therefore, some
efforts have been made to replace chemical surfactants
by BS in the development of metal NP.
Surfactin was successfully applied as a stabilizing
agent in the formulation of silver (AgNPs) and gold NPs
and their stability, size, shape, and polydispersity were
dependent on the pH and temperature [43,44]. A glyco-
lipid from Brevibacterium casei MSA19 was utilized to
prepare AgNPs by the microemulsion-based technique
using reverse micelles. The authors evidenced that NPs
were stable for 2 months and postulated that BSs acted
as stabilizer preventing the formation of aggregates
[45]. Spherical AgNPs were obtained using NaBH4 as
reducing agents in BSs reverse micelles further
extracted into heptane. The Pseudomonas aeruginosa
BS favored the synthesis of AgNPs stable for 3 months
without passivator addition [46].
BSs also play the role of both capping and reducing
agents in NP synthesis.
A lipopeptide surfactant produced by Bacillus subtilis
ANR 88 using molasses as the carbon source was uti-
lized for the synthesis of gold and silver NPs. The
AgNPs showed spherical shape in the size range of
4–18 nm, whereas gold NP was mostly hexagonal from
40 to 60 nm size. Authors conclude that the lipopeptide
was effective to stabilize and permits the synthesis of
the NP in the absence of chemical reducing agents
being a potential candidate for green NP preparation
[47]. A BS extracted from corn steep liquor, spontan-
eously fermented by lactic acid bacteria, was applied to
the synthesis of metal NPs using a one-step procedure
induced by temperature. A mixture of gold nano-
spheres and nanoplates was observed after the reduc-
tion of gold precursors induced by BS. Additionally,
AgNP of pseudo-spherical shape were also obtained.
The analysis of BS revealed the lipopeptide nature and,

according to the authors, the BS was essential to the
reduction as well to the stabilization of the NP [48]. A
simple and green method for the synthesis of gold NPs
using the glycolipid surfactant mannosylerythritol lipid
(MEL) produced by Ustilago maydis CGMCC 5.203 was
proposed [49]. The MEL was utilized as a green reduc-
ing/stabilizing agent generating spherical and uniform
NP with potential bioactivity. Recently, a one pot
greener synthesis of gold NP was proposed using soph-
orolipid as a reducing and capping agent resulting in
gold NP with potential application in biomedicine [50].
Most studies regarding the use of BS involve the syn-
thesis of silver and gold NP since those metals have
well-known biological activity and low toxicity; and, the
combination of such NP with BS can improve their bio-
activity. However, BSs were also utilized for the synthe-
sis of NP of cobalt [51], cadmium sulfide [52], nickel
oxide [53], zinc sulfide [54], zinc oxide [55], and iron
oxide [56].
Some attempts to obtain metal NP by extracellular
biogenic synthesis using BS-producing microbes were
also reported. In this case, microbial growth is per-
formed and the resulting culture medium is separated
from cells and utilized as starting material for NP syn-
thesis after the addition of a metal precursor. Krishnan
et al. [57] described the synthesis of cubic shape AgNP
with an average size of 15.40 nm using culture filtrates
of a surfactin producing Brevibacillus brevis. The BS was
responsible for the reduction and stabilization of the
resulting NP, which exhibited potential for the treat-
ment of Gram-negative infection in wounds. Green syn-
thesis approach using a B. subtilis culture broth and
microwave radiation resulted in AgNP with potential to
encapsulate surfactin synthetase ligands useful for drug
design and development [58]. The properties of AgNP
obtained by the reduction of silver nitrate using bio-
logical and chemical methods in the presence of a B.
subtilis surfactant have been compared. The presence
of surfactin significantly increases the stability of the
AgNP in both bio and chemical protocols [59].
Organic nanoparticles
Although considerable research regarding the green
synthesis of metal NPs are available, their application in
organic NP synthesis is an emerging field. Different
from metal NP, where BSs are primary applied as stabi-
lizing and/or reducing agents, in organic NPs, BSs are
usually functional agents to improve biological activity.
Interesting work conducted by Hazra et al. [60] pro-
posed the use of BS to replace SDS in the green synthe-
sis of poly (methyl methacrylate) (PMAA) NPs. Using an
atomized microemulsion technique, the authors
CRITICAL REVIEWS IN BIOTECHNOLOGY 5
synthesized nPMMA (core) – BS (shell) particles with
surfactin, RLs, and trehalose-lipids. The resulting
20–50 nm diameter spherical shape NPs were nontoxic,
biocompatible, exhibited strong antibacterial activity
against B. subtilis and P. aeruginosa, and exhibited
potential as drug delivery systems. These studies
pointed out BS-polymer hybrids as the next-generation
delivery vehicles for biomedical applications. Surfactin-
loaded polyvinyl alcohol (PVA) nanofibers were
obtained by gravity electrospinning. The nanofibers
containing surfactin were thinner and with lower crys-
tallinity than nanofiber with pure PVA. Surfactin-loaded
nanofibers prevented the adhesion of bacterial cells to
polystyrene and that could be applied to prevent bio-
film formation in prosthetic devices [61]. Gelatin nano-
fibers containing a lipopeptide BS were fabricated by
electrospinning techniques. The percentage of water
uptake by the surfactin nanofibers, one of the most
essential factors for wound healing and covering mate-
rials, was around 2.5 times higher than the commer-
cially available ones [62]. More recently, our group
reported the formulation of a biopolymer-BS NP useful
for biomedical applications. The combination of chito-
san and RLs generated stable and monodispersed NP,
spherical in shape with an average size of around
288 nm and positively charged. The addition of RL
reduced the size and polydispersity index of chitosan
NP and improved the antimicrobial activity against
Staphylococcus aureus even planktonic or biofilms. The
low cytotoxicity and high antimicrobial potential dem-
onstrated by the chitosan-RL NPs suggest that they can
be exploited to the design of novel strategies to control
S. aureus in pharmaceutical and food industries [63].
In a similar study, Bettencourt et al. [64] proposed
the preparation of chitosan-based NP as carriers for
antimicrobial glycolipids. Stable NPs with an encapsula-
tion efficiency of 41.1% and 74.2% and average size of
210.0 and 329.6 nm were obtained with sophorolipids
and RLs, respectively. The RL-chitosan NP has better
antimicrobial activity against S. aureus than sophoroli-
pid-chitosan NP. Authors attributed the reduced effects
of the latter to the hiding of the positively charged
groups or reducing charge density of biopolymer
caused by the inclusion of TweenV R 80 surfactant in the
formulation. In conclusion, the synergy among chito-
san-BS is dependent on the kind of BS/biopolymer and
the formulation system utilized.
Biosurfactant self-assembly structures
The ability of amphiphilic molecules to spontaneously
self-assemble in solution results in a variety of
6 M. NITSCHKE AND C. A. MARANGON
Figure 2. Effects promoted by BS nano-sized structures in cell membrane.
supramolecular structures. BS micelles, lamellas, vesicles
[65], and several other forms such as sponges and lyo-
tropic liquid crystals [66] have been described. The self-
aggregation of BS is dependent on pH, temperature,
and ionic strength and this property is a useful tool for
the preparation of smart drug delivery systems [67].
Besides, the similarity of some BS molecular aggregates
with cell membrane organization favors their fusion
and uptake (Figure 2).
Nanomicelles were prepared by sonicating surfactin
in distilled water and PBS buffer for 15 min at 30  C.
The nanomicelles formed in water were spherical in
shape and smaller in size than those formed in PBS
where larger and amorphous aggregates were
observed and authors attribute the difference to the
ionic strength of the PBS buffer. The resulting nanomi-
celles inhibited the growth of tumoral cell lines (HeLa)
in a time and dose-dependent mode [68]. The potential
of RLs as nanocarriers to drug delivery systems was
investigated. The RLs formed spherical structures with
average sizes ranging from 5 to 100 nm, low polydis-
persity, and stability over a wide concentration range.
The authors demonstrated the size of the nanostruc-
tures depends on the length of the hydroxyl-fatty acids
component of RL, which in turn determines the packing
parameters of the BS. The nanocarriers were nontoxic
to human fibroblasts and good candidates for the der-
mal delivery of drugs [69]. Curcumin was successfully
entrapped in micelles of acidic sophorolipids. Stable
micelles with ellipsoidal shape and average size around
100 nm were formed. According to the authors, the
presence of the BS increased the solubility of hydropho-
bic curcumin molecules enhancing their fluorescence
property. The uptake of such micelles was demon-
strated in both Escherichia coli and S. aureus also, a
quenching activity against P. aeruginosa was observed.
The fluorescence presented by curcumin-sophorolipid
micelles permits their use as biomarkers in confocal
microscopy and theranostic applications [70]. Similarly,
curcumin was loaded in sophorolipid micelles and NPs
were formed by changing the pH to decrease the curcu-
min solubility. The sophorolipid-curcumin particles had
around 61 nm in size and a negative charge. The coating
of curcumin with sophorolipid increased the bioavailabil-
ity, owing to their ability to increase the solubilization of
the curcumin in the mixed micelle phase [71].
Two recent patents claim the preparation of RL lipo-
somes loaded with bioactive peptides to the develop-
ment of antimicrobial products for use in medicine [72]
and agriculture [73]. RL functionalized liposomes (rham-
nosomes) were prepared and loaded with a bacteriocin.
The addition of RL increased the encapsulation effi-
ciency of nisin, the size, and the physical stability of the
nano-vesicles. The rhamnosomes demonstrated higher
antimicrobial activity than liposomes and enhanced the
activity of nisin against Gram-positive and Gram-nega-
tive resistant foodborne pathogens [74]. More recently,
the rhamnosomes were incorporated in a biopolymer
composite to develop nano-active packing for food.
The coating exhibited broad-spectrum antimicrobial

activity against Listeria monocytogenes and E. coli repre-
senting a new strategy to improve food preserva-
tion [75].
BS in nanoemulsions
Emulsions are applied in a wide range of industrial fields
such as cosmetics, pharmaceuticals, food, chemicals, tex-
tiles, and petroleum [76]. Nanoemulsions are kinetically
stable dispersions of two immiscible liquids, stabilized by
a surfactant, with droplets size lower than 200 nm
[76,77]. Because of their smaller size, nanoemulsions
show advantages over conventional emulsions such as
higher physical stability, transparent appearance, tunable
rheology, and increased bioavailability [78,79]. There is
increasing interest in developing long-term stable nanoe-
mulsion using sustainable surfactants [80,81], which can
be applied as vehicles to hydrophobic compounds, such
as essential oils in cosmetics, pharmaceuticals, and foods
[82]. BS can replace synthetic surfactants in nanoemul-
sions formulation or/and act as an active or synergic
agent once they present diverse biological properties.
Although microbial surfactants are good candidates for
nanoemulsion preparation, few reports are available in
the literature. Joe et al. [83] described the development
of surfactin-based nanoemulsions using cooking oils.
Surfactin–sunflower oil nanoemulsion showed the lowest
particle size (72.52 nm) and was selected to further anti-
microbial studies. The formulation inhibited the growth
of Salmonella typhi, L. monocytogenes, and S. aureus.
Fungicidal activity against Rhizopus nigricans, Aspergillus
niger, and Penicillium sp., and sporicidal activity against
Bacillus cereus and Bacillus circulans were observed. The
antimicrobial potential of the nanoemulsion was also
evaluated in food models. Authors report a significant
reduction in the native bacterial and fungal populations
present in raw chicken, apple juice, milk, and mixed vege-
table. The surfactin-based nanoemulsion showed
improved antimicrobial activity and potential for the
development of preservatives for food applications.
Bai and McClements [81] studied the physicochemi-
cal properties of nanoemulsions of RL and saponin with
medium-chain triglyceride (MCT), fish oil, corn oil, and
lemon oil. Authors conclude that RLs have similar inter-
facial properties as the commercially available natural
surfactant (quillaja saponin). RLs were able to form
nanoemulsions with small droplet diameters with the
different oil types. The RL-coated droplets were stable
to aggregation over a range of pH (5–9), salt concentra-
tions (<100 mM NaCl), and temperatures (2090  C)
showing potential for further applications. Formulation
of a surfactin-stabilized nanoemulsion designed to
CRITICAL REVIEWS IN BIOTECHNOLOGY 7
encapsulate active compounds such as vitamin C, E,
and curcumin was recently reported. The nano-sized
delivery system was stable for 6 months and showed
improved bioavailability and distribution of the actives
through the skin suggesting good potential for cos-
metic and dermatological applications [84].
Other BS application examples
Polyaniline (PANI) is an electrical conductive polymer useful
for a vast range of technological applications. PANI NPs
were synthesized using RLs as templates. The BS template
generated PANI nanotubes and nanorods with uniform
size, morphology, and higher crystallinity and electrical con-
ductivity than those obtained without the BS. According to
the authors, the utilization of RLs as a soft template showed
advantages over synthetic surfactants, once they can be
readily removed under mild conditions [85].
Nanodiscs are membrane models composed of a
phospholipid bilayer held together by a membrane
scaffold protein (MSP). In this view, RLs were utilized to
prepare nanodiscs using the MSP1D1. The formation of
rhamnodiscs of di-RL and MSP1D1 was demonstrated
showing strong dependence on BS: protein molar ratio
and such nanostructures can represent a novel insight
to research in biomimetic membranes [86].
A new method for the design of small polymer par-
ticles utilized as a reference in toxicity studies of pollu-
tion caused by micro and NPs of plastics in the aquatic
environment was recently proposed. Polyethylene (PE)
micro and nanoplastics were obtained using Tween 60,
Tween 80, and a BS from the algae Chlamydomonas
reinhardtii. Using the BS, the presence of an eco-corona
at the surface of the particles was mimicked, improving
their uptake by Daphnia magna. The obtained particles
can be used as model microplastics to evaluate the
eco-toxicity of PE [87].
The BS can play multiple roles in nanotechnology: in
the synthesis of NP they act as capping and/or reducing
agents; they also provide self-assembly structures to
encapsulation, functionalization, or templates and act
as emulsifiers in nanoemulsions. Owing to their intrinsic
biological properties, BS can also play as active com-
pounds and their inclusion in nanostructures affects not
only the physicochemical character but also biological
activity thereby, their range of applications (Table 2).
Impact of BS in biological activity of
nanostructure materials
As discussed above, besides surface activity, one of the
most important features demonstrated by BSs is their
8 M. NITSCHKE AND C. A. MARANGON
biological activity. Antimicrobial, anticancer, anti-adhesive,
antibiofilm, immunosuppressive, immunomodulating,
anti-inflammatory, and antioxidant properties have been
described and reviewed in the literature [27,92,93]. These
properties can also be exploited in nano-sized materials.
Biomedical applications
Antimicrobial activity
BSs are able to inhibit the growth of several pathogenic
and environmental strains of bacteria, fungi, yeasts,
algae, and viruses. Although the exact mechanism is
unclear, the antimicrobial action of BS is associated
with their amphiphilic character, which allows them to
disturb the cell membrane (Figure 2), leading to
increased permeability, pore formation, metabolite leak-
age, and cell lysis [94,95]. The antimicrobial activity of
BS nanostructures combination is, in general, attributed
to the greater binding ability to the cell surface pro-
moted by the surfactant. Moreover, BS particular activ-
ity against several microbes helps to improve the
antimicrobial action of nanostructures in a synergis-
tic mode.
Chitosan-RL NPs were more effective than both RL
and chitosan NPs , against planktonic and biofilms of S.
aureus. Authors conclude that the high local density of
polycationic chitosan in the hybrid NPs leads to greater
electrostatic interactions and the release of RL close to
the bacterial cell surface (Figure 3), which aids disrup-
tion of the cell envelope and subsequent access of anti-
microbials to their cell targets [63].
An acidic sophorolipid diacetate synthesized by a
Cryptococcus sp. strain was employed to develop bio-
functionalized zinc oxide NP able to inhibit the growth
of the pathogens Salmonella enterica and Candida albi-
cans. The enhanced antimicrobial activity promoted by
the sophorolipid BS was associated with increased cell
permeability and protein leakage. FTIR analysis of
pathogens before and after treatment with the NPs
indicated amino, thiol, and carbonyl groups as predom-
inant contributors to the binding of functionalized NP
to the cells. Furthermore, the presence of an increasing
number of thiol groups in the cell wall of S. enterica
was correlated to higher activity demonstrated against
the bacterium compared to the yeast [55].
Antimicrobial potential of gold NP functionalized
with sophorolipid was recently reported [50]. The
uncapped gold NP did not exhibit antibacterial action,
however, their combination with the BS significantly
improved bacterial inhibition with higher efficacy
against Gram-negative bacteria such as Vibrio cholerae.
The sophorolipid mediated the disturbance of cell
membrane and pore formation helping the NP enter
the cell and binding to respiratory enzymes as LDH,
blocking its activity. The gold-SL NPs demonstrated syn-
ergic action with polymyxin and kanamycin showing
potential for antimicrobial therapy in nanomedicine
[50]. A nonspecific synergistic effect of AgNPs in com-
bination with BS was also reported. The BS produced
by a B. subtilis strain increased the stability of biogenic
AgNP and enhanced their antimicrobial activity against
phytopathogenic fungi and Gram-positive bacteria. In
the presence of BS, the mycelium growth rate inhibition
was around 50%, while no effect was observed for
chemical synthesized AgNP (without BS) against most
fungal strains. To study the mechanism(s) involved in
antimicrobial activity, the conjugation of bacterial DNA
and the AgNP was observed. The DNA accumulated
more densely around biogenic NP suggesting a strong
affinity between metal NP and chemical groups of DNA.
The presence of BS probably favors NP-DNA interaction
by increasing affinity to the cell surface and NP
uptake [96].
The activity of BS, especially against enveloped virus,
is well-known and documented in the literature [97,98]
although they are not exploited commercially to such
an application. This scenario may be modified by the
emergence of coronavirus (COVID-19) pandemic disease
which opened new opportunities to BS. In this sense,
some proposals to combat COVID were recently
reported. Therapeutic formulations can include BS to
help the emulsification of lung fluids with the conse-
quent improvement of patient’s ventilation in an acute
respiratory syndrome [99]. Another study hypothesized
that potential anti-inflammatory activity presented by
BS may reduce the cytokine storm and inflammation in
COVID patients [100]. BS are capable of inactivate virus
by solubilizing and disrupting the lipid envelope or by
targeting capsid proteins [97,98]. Thus, as COVID-19 has
an envelope and also capsid and surface spike proteins
it is expected that such pathogens can also be suscep-
tible to BS-based treatments [100,101]. Development of
BS:NPs, micelles, nanoemulsions, and other nanostruc-
tures taking advantages of both “nano” properties and
BS activity; even individually or combined with other
bioactive compounds, may improve the delivery and
efficiency of proposed COVID-19 treatments. For
example, aerosol forms using BS-nano-sized delivery
systems can be applied to directly reach the respiratory
tract. Nevertheless, it is imperative to perform “in vitro”
and “in vivo” clinical experiments to confirm the effect-
iveness of the proposed strategies. Toxicity tests, side
effects, and mechanisms involved in BSs and their
nano-sized structures action must be determined.
 CRITICAL REVIEWS IN BIOTECHNOLOGY 9

Table 2. Role of BS in the formulation of nano-sized structures and their applications.

BS type Nanostructure type BS function Proposed application Reaction conditions References
Glycolipid Silver NP Stabilizing Green synthesis 0.1 g/L BS (n-heptane) þ1 mL AgNO3/ [46]
inorganic NP stir.10 min. room temp.þ 1 mL
NaBH4/30 min. Further extraction
with 10 mL ethanol and
centrifugation.
MEL Gold NP Stabilizing Anticancer, 200 lL MEL (MeOH/ H2O 1:10) þ [49]
and reducing antioxidant 50 mL 3 mM HAuCl4 stir. 60  C –
30 min, pH 8.
Sophorolipids Gold NP Stabilizing Antimicrobial 40 lL SL (100 mg/mL) þ 10 mL [50]
and reducing HAuCl4 (400 lg/mL) þ few drops
NaBH4 (100 mM) pH 5.5 80  C
Surfactin NP Nanocarrier Anticancer Doxorubicin HCl (1.0 mg/mL) þ [88]
triethylamine (15 lL/mL) in CHCl3
40 min room temp. Drop into
Surfactin (2 mg/mL) þ ultrasonic
emulsification 5 min., stir.
40  C overnight.
Surfactin, rhamnolipids Organic (PMMA) NP Green Drug delivery, Patented atomized microemulsion [60]
and trehalose lipids emulsifiers, active antimicrobial technique. BS þ water, 200 rpm/
55  C. Add ammonium persulfate
follow methyl methacrylate
spraying. After 1 h at 55  C PMMA
NP extracted from the latex
with methanol
Rhamnolipids Organic (chitosan) NP Active Antimicrobial, Depolymerized chitosan (0.5 mg/mL in [63]
antibiofilm acetic acid) þ RL (0.5 mg/mL)
mixed 1:1 under stir. 700 rpm,
25  C/ 24 h. 3 mL sodium
tripolyphosphate (0.5 mg/mL)
added drop wise at 700 rpm.
Surfactin Organic (PMMA NP Nanoadsorbent Toxic metal removal Sonochemical emulsion [89]
polymerization. Aqueous solutions
methyl methacrylate (20%) þ
potassium persulfate (0.4 0%) þ
surfactin (4.0%) , 55  C/1 h. Power
output, 50% amplitude.
Frequency 22 kHz.
Surfactin Nanofibers Active Antibiofilm, Aqueous Polyvinyl alcohol 10% (w/v), [61]
anti-adhesive 85  C/1 h vortex. Sodium surfactin
solution (0.5–1.5%) was added at
28 C. Gravity electrospinning
technique under constant 12.5 kV,
collector plate charged at 5 kV,
relative humidity 4050%.
Lipopeptide Nanofibers Active Wound healing Gelatin solution 20% (w/w) þ BS [62]
(25%) mixing 3 h. Electrospinning
technique with 6 kV voltage, flow
rate, 0.5 mL /h30 min and 10 cm
distance of needle tip and
fiber collector.
Sophorolipid Micelles Encapsulation Biomarker, theranostic Curcumin (1 mg/mL) probe-sonicated [70]
for 40 min with acidic SL
solution (5%)
Rhamnolipid Liposomes Nanocarrier Antimicrobial 20 mg nisinþ 50 mg of [75]
active packing phospholipids þ RL (3:1 w/w) in
20 mL PBS pH 7. Heat 35  C ,
homogenized at 13,500 rpm, and
ultra-sonicated 15 min at 25 kHz.
Nanocarrier add to 1:1 (v/v) blend
j-carrageenan/ hydroxypropyl
methylcellulose.
Surfactin Nanoemulsions Emulsifier/active Antimicrobial Cooking oil (14%) þ ethanol (3%) þ [83]
Surfactin 20% (v/v) mixed, left 1 h/
86  C. Oil phase mixed with water
and centrifuged 10,000 g.
Rhamnolipid Nanoemulsions Emulsifier Cooking oil emulsion 10% Oil þ 90% aqueous RL (0.1–3.0% [81]
in 10 mM sodium phosphate, pH
7.0). Microfluidizer emulsions
obtained under different
homogenization pressures
(9–19 kpsi).
(continued)
10 M. NITSCHKE AND C. A. MARANGON

Table 2. Continued.
BS type Nanostructure type BS function Proposed application Reaction conditions References
MEL- A Liposomes Stabilizing/active Anticancer Soy phosphatidylcholine-cholesterol [90]
3:1 þ 10% MELþ 1% betulinic acid
all solved in chloroform and
evaporated 40  C/150 rpm. Lipid
film dispersed into PBS by
ultrasonic cleaner 15 min, follow
3 min in ultrasonic cell grinder.
C. parapsilosis BS Graphene quantum dots Active/bioconjugate Cancer diagnosis 25 mL of GQDs (85 mg/mL in PBS, pH [91]
and therapy 7.4) þ EDC (2 mg) þ NHS (3 mg)
at room temp./1 h. 300 lL BS
(1 mg/mL in PBS, pH 7.4)
conjugated to GQDs through
amine-carboxyl coupling reaction
1 h at RT under stirring. Then 3 mL
of BS-GQDs mixed with 30 lL of
Folic acid (1 mM PBS) 1 h stirring
and centrifugation
(6000 rpm/15 min)
Rhamnolipid Nanodiscs Self-assembly Biomimetic RL dissolved in 200 mM sodium [86]
with protein membranes cholate sonicated 10 min, 37 Hz
/30  C. Membrane scaffold protein
added to different ratios and
heated 130  C/1 h following
dialysis removal of sodium cholate
to self-assembly of nanodiscs
NPs: nanoparticles; BS: biosurfactant; PMMA: poly methyl methacrylate; MEL: mannosyl erythritol lipid; GQDs: graphene quantum dots; EDC: N-(3 dimethy-
laminopropyl)-N-ethylcarbodiimidehydrochloride; NHS: N-hydroxysuccinimide.

Another interesting approach to exploit BS-based
nanostructures is the development of antiviral protect-
ive clothes, masks, gloves, and coating films to minim-
ize the lifespan of viral particles in surfaces and reduce
their transmission. Antiviral adhesive coatings or cloth
fabric loaded with hybrid NPs (metal-BS, chitosan-BS) is
a more feasible strategy to apply BS-nano-sized struc-
tures in the short-term, once their development does
not demand extensive clinical studies.
Anti-biofilm activity
Biofilms are defined as a microbial community sur-
rounded by an extracellular polymeric matrix, which is
attached to biotic or abiotic surfaces [102]. Compared
to planktonic, the sessile lifestyle offers greater resisting
power to physical and chemical agents since the matrix
acts as a protective barrier that prevents contact with
antimicrobials [103]. Biofilm-related infections are a
matter of concern once recalcitrance toward antibiotics
can lead to treatment failure and infection recurrence
[104]. Microbial adhesion to surfaces is the first step of
biofilm formation hence, an interesting strategy to con-
trol biofilms is to prevent adhesion. BS are able to mod-
ify or to form a conditioning film over abiotic surfaces
resulting in a delay or reduction of microbial adhesion
and formation of biofilms [105]. These anti-adhesive
properties are relative to changes in the electrostatic
and/or hydrophobic interactions between bacteria and
surface promoted by the presence of BS. If repulsive
forces dominate, cell adhesion can be avoided [106].
Once a biofilm is established, BS can act as a disrupt-
ing agent. Biofilm removal involves the weakening of
biofilm structure due to reducing surface and interfacial
tensions, thus favoring dispersion and disintegration of
the matrix [107,108]. NPs have also been applied to the
treatment of biofilms as vehicles to active antimicrobial
molecules. Generally, NP are surface-functionalized or
loaded with drugs to facilitate their penetration and
favor the controlled release inside the biofilm matrix
[109]. Reports in the literature regarding BS-based
nanostructures on controlling biofilms are scarce. The
presence of RL in chitosan NP helps the penetration
and release of RL to the lower layers of S. aureus bio-
films (Figure 3), suggesting that the BS can be exploited
to the design of novel anti-biofilm nanostruc-
tures [63,64].
Anticancer activity
The antitumoral activity of BS is also well recognized.
Most studies concerning anticancer effects of BS
include the lipopeptide class. A review from Wu et al.
[110] emphasizes the potential of surfactin against sev-
eral types of tumors such as Ehrlich ascites carcinoma,
breast cancer, colon cancer, leukemia, hepatocellular
carcinoma, and cervical cancer. According to the
authors, induction of apoptosis and cell growth inhib-
ition are the main mechanisms reported to be

Figure 3. Conceptual image of the interaction of chitosan-rhamnolipid NP with S. aureus cell wall and biofilms.
responsible for the anticancer activity of surfactin.
However, cell cycle arrest, metastasis inhibition, oxida-
tive stress, and mitochondrial damage have also been
associated with tumor anti-proliferative effects of BS
[90,110,111]. Due to their small size, nanostructures can
improve the efficacy of anticancer treatments once they
can increase drug accumulation in the tumor by
enhancing the permeation and retention effect
[88,110]. In this view, studies aiming to improve anti-
cancer action of BS by combining them with nanostruc-
tures, have been described in the literature. RL-
functionalized AgNPs effectively inhibit in vitro prolifer-
ation of human breast adenocarcinoma cells (MCF-7)
comparatively to normal lymphocytes cells (PBMN). The
difference observed in cell viability was associated with
more selective binding of the anionic NP to the tumor
cell surface, which in turn, lead to membrane disruption
and oxidative stress [111].
MEL-A BS produced by a Pseudozyma aphidis strain
was utilized together with betulinic acid to the formula-
tion of liposomes with anticancer activity. Liposome for-
mulation containing soy lecithin-cholesterol (3:1), 10%
MEL-A, and 1% betulinic acid was tested against human
hepatoma cells (HepG2). The incorporation of MEL-A
CRITICAL REVIEWS IN BIOTECHNOLOGY 11
resulted in uniform particle size distribution and
improved stability of liposome particles. Furthermore,
MEL-A improved cell apoptosis and the destruction of
mitochondrial membrane potential in HepG2 cells. The
presence MEL-A facilitated the permeation and delivery
of the drug and synergistically enhanced the anticancer
activity of betulinic acid liposomes [103].
A surfactin-based NP-loaded with the anticancer
drug doxorubicin was synthesized by solvent-emulsion
method generating a core–shell structure. The NP
showed stronger cytotoxicity against doxorubicin-resist-
ant human breast cancer cells (MCF-7/ADR) compared
to free doxorubicin. Enhanced cellular uptake and
decreased cell efflux were observed when tumor cells
were treated with the NP. A superior tumor inhibition
with fewer side effects was observed using in vivo
model. In summary, surfactin nanocarriers may repre-
sent a promising alternative to overcome multidrug
resistance in cancer chemotherapy [88].
Advantageous properties of graphene quantum dots
(GQDs) such as stable photoluminescence, low cytotox-
icity, and high biocompatibility attracted great atten-
tion in many fields, giving them the status of next-
generation nanomaterials [112]. Bansal et al. [91]
12 M. NITSCHKE AND C. A. MARANGON
proposed the preparation of GQDs conjugated with a
BS produced by Candida parapsilosis. The bioconjugate
was characterized as round-shaped homogenous dis-
persed structures with a 3.58 nm size range.
Conjugation of GQDs with BS reduced significantly the
viability of human breast cancer (MCF-7) cells, and fur-
ther decoration of the BS-GQDs conjugate with folic
acid enhanced their cell uptake and antitumoral activ-
ity. The photoluminescence of bioconjugated GQDs
was adequate for targeting and imaging the cells thus,
they have potential application in cancer diagnosis
and therapy.
Green synthesized gold NP obtained using MEL from
U. maydis demonstrated a potential cytotoxic effect
against human liver hepatoma (HepG2) cells. Also,
DPPH and ABTS radical scavenging measurements con-
firmed their antioxidant properties and the NP exhib-
ited antimicrobial activity against S. aureus and E. coli.
The presence of MEL played an essential role to
develop metal NP for biomedical applications [49].
Most studies focusing on the medical application of
BS-based nanostructures evaluate in vitro toxicity, how-
ever, in vivo toxicity and the environmental impact of
such structures are not emphasized. This gap should be
properly covered by future studies.
Environmental applications
Despite BS has been traditionally applied for environ-
mental purposes, the development of BS-based nano-
structures for such applications is scarce. A unique
example regarding environmental application of BS-NP
was reported by Kundu et al. [89] which described the
synthesis of poly (methyl methacrylate)-surfactin NP by
ultrasound-assisted emulsion polymerization. The NPs
were efficient nano-adsorbent to the removal of heavy
metals from water. The metal ions adsorption mechan-
ism involves the formation of chelates or electrostatic
interactions with NP showing selective affinity to Co2þ
> Zn2þ > Ni2þ > Cr3þ >Fe2þ > Cu2þ > Cd2þ > Pb2þ
respectively, under single-component conditions. The
NP preserved four adsorption–desorption cycles with-
out significant loss of adsorption capacity. The surfac-
tin-functionalized NP was also able to remove phenolic
compounds, exhibited antimicrobial activity against E.
coli, and low cytotoxicity showing potential as an envir-
onmental-friendly alternative to the treatment of waste-
water [89].
Few studies explore the combination of NPs and BS
to improve bioremediation of oil. In those examples, BS
was not utilized in the synthesis of NPs, but are mixed
with them to enhance their action in a synergistic
mode. The small size of NP permits their flow through
pores in oil reservoirs and the presence of BS helps to
improve the recovery processes [113]. Adsorption of
emulsan BS into SiO2 NP forms a BS monolayer at the
particle surface making them more hydrophobic and,
enhancing the recovery of oil [113].
Silica NP and RLs were able to form and stabilize
emulsions of tetradecane or crude oil in water and sea-
water. The NP and BS acted synergistically generating
emulsions with smaller oil droplets and greater stability
to coalescence than emulsion stabilized by either NP or
BS alone. Highly stable crude oil-seawater emulsions
were obtained with 2% silica NP þ 60 mg/L RL. The
environmental-friendly combination of silica and RLs
showed potential to be applied in the remediation of
oil spills [114]. In a similar study, oil recovery by simul-
taneous flooding of silica NP and RLs was investigated.
A synergistic effect between BS and NP resulted in
enhancing oil recovery favoring the release of trapped
oil. Authors propose that the reduction of interfacial
tension promoted by BS lowered capillary forces help-
ing the diffusion of NP and BS molecules through the
pores, which in turn, modifies the wettability. In add-
ition, the increase in viscosity of injected fluids pro-
moted by the NP and BS was responsible for the
enhanced oil recovery observed [115].
Apart from the countless advantages and potential
applications presented by nano-sized materials, their
emergence gave rise to concerns. In fact, like any new
technology, questions about the impact of nanomateri-
als on health and the environment are still waiting for
answers. The smallest size and highest surface area,
which can improve distribution in living organisms, are
key advantages of nano-sized structures; on the other
hand, these features are also associated with improved
toxicity. Inhalation of NPs was related to pulmonary
problems. Moreover, dermal, nephron, and hepatotox-
icity of some nano structures have been
reported [116,117].
Nano-sized materials are found in natural environ-
ments such as water, soil, and air. This fact might be
associated with their greater stability compared to bulk
counterparts together with increasing commercial
exploitation, making them prospective candidates to
pollutants in a short time [118]. Therefore, the develop-
ment of standardized and validated methods for quan-
tification and identification of nano-sized materials in
the environment is increasingly necessary for their safe
utilization [119].
It is important to emphasize that although BS are, in
general, considered to be low toxic and environmental
friendly, their formulations at nanoscale (especially

when combined with other materials) cannot simply be
assumed to be safe. To reach successful applications in
nanotechnology, the issues relative to health and envir-
onment regarding BS-based nanostructures should also
be addressed.
Conclusions and perspectives
BSs have emerged as green alternatives to the formula-
tion of nano-sized structures since their physicochemi-
cal, self-assembly, and biological properties, combined
with their natural status, are advantageous over syn-
thetic surfactants. The use of BS in both, inorganic and
organic (polymeric) NPs , nanoemulsions, micelles, and
liposomes were proposed in the last years to explore
their properties not only as capping, reducing agents,
emulsifiers and nanocarriers but also as active agents.
As the economics of some BS production is becom-
ing feasible, the traditional concerns about their relative
higher production costs are replaced by the lack of
robust information about their oral toxicity and impact
on human microbiota/microbiome, especially for
pharmaceutical and food applications. Besides, the
awareness about health and environmental impact of
the disseminated use of nano-sized materials demands
more research to fulfill safety and regulatory issues. By
contrast, several studies show that when BS are com-
bined in nano-sized structures, their in vitro cytotoxicity
decreases, representing an additional feature to boost
their use in nanotechnology.
Most applied research of BS-based nanostructures
focuses on biomedical areas where they improve or act
synergistically with other biological-active molecules as
antimicrobials and anticancer as well for diagnostic pur-
poses. Efforts to elucidate the mechanisms involved in
BS-cell interaction are needed to improve the effective-
ness of BS in nanomedicine. Tailor-made BS is an
emerging trend that can solve the problems relative to
the lack of uniformity in composition presented by nat-
ural BS. Improved activity and reduced toxicity are
some characteristics that can be designed for spe-
cific needs.
Considering the potentiality demonstrated by BS,
their applications in nanotechnology are far from being
exhausted. The development of biosensors for diagno-
sis, antimicrobial and active coatings, and intelligent
nanomaterials are some potentially unexplored trends
in the health sector. Environmental, agriculture, cosmet-
ics, oral care, and household detergents are other
examples of currently innovative sectors for BS-nano-
sized structures. Specifically for the environment and
agriculture, the control of plant pathogens or insects
CRITICAL REVIEWS IN BIOTECHNOLOGY 13
and the remediation of pollutants are prospective, since
in such applications, there are no concerns about their
oral intake safety. In conclusion, we can expect an
increasing demand for the development of BS nano-
sized products in the forthcoming future.
Disclosure statement
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this article.
Funding
Authors thank to Fundaç~ao de Amparo a Pesquisa do Estado
de S~ao Paulo (FAPESP) Grant n 2019/14405-9 for finan-
cial support.
ORCID
Marcia Nitschke http://orcid.org/0000-0001-6107-4183
Crisiane Aparecida Marangon http://orcid.org/0000-0002-
8499-6735
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