Microbial

pathogens and strategies for combating them: science, technology and education (A. Mndez-Vilas, Ed.)
____________________________________________________________________________________________

Marine microbial biosurfactants: Biological functions and physical

properties as the basis for innovations to prevent and treat infectious
diseases in aquaculture
M. A. Dinamarca1, *, C. J. Ibacache-Quiroga1,3, J. R. Ojeda1 and J. M. Troncoso2
1

Laboratorio de Biotecnologa Microbiana, Escuela de Nutricin, Facultad de Farmacia, Universidad de Valparaso, Gran
Bretaa 1093, 2360102, Valparaso, Chile
2
EWOS Chile Alimentos Ltda, Coronel, Chile
3
Centro Nacional de Biotecnologa, Consejo Superior de Investigaciones Cientficas (CSIC), Universidad Autnoma de
Madrid, Darwin 3, 28049, Madrid, Spain
*Corresponding author: M. A. Dinamarca, Laboratorio de Biotecnologa Microbiana, Escuela de Nutricin, Facultad de
Farmacia, Universidad de Valparaso, Gran Bretaa 1093, 2360102, Valparaso, Chile
Aquaculture is a rapidly growing economic area that represents an important alternative to satisfy future food demands.
Nevertheless, one of the main threats to aquaculture is infectious outbreaks that generate significant productive losses. In
response, large quantities of antimicrobials have been used and released into aquatic ecosystems, with the associated risk
to human health and the environment, making it necessary to develop new products that are safe for humans and the
environment. This chapter focuses on the use of marine microbial biosurfactants, a wide variety of compounds with
remarkable physical and biological properties, as an alternative to antibiotic therapy in aquaculture.
Keywords biosurfactants; antimicrobials; fish pathogens

1. Introduction
The overexploitation of marine resources, combined with climate change, is reducing marine resources as a food source.
In this scenario, aquaculture is a rapidly growing activity that offers an important alternative to satisfy future food
demands. According to the Food and Agriculture Organization of the United Nations (FAO), food resources produced
by aquaculture have increased 12 times in recent decades, at an average annual rate of 8.8 percent, with about 600
different species, and 190 countries involved in farming systems [1]. The main threats to this activity are infectious
diseases that can cause total losses to farmers. In order to maintain growth rates, large quantities and varieties of
antimicrobial agents have been used in aquaculture, including orally dispensed antibiotics in fish feed. The worldwideapproved antimicrobials agents for aquaculture include a variety of aminoglycosides, beta-lactams, macrolides,
quinolones sulfonamides and tetracyclines that are also employed for prophylaxis and treatment of infections in
humans. These drugs are applied under specific regulations established by the respective health agencies of each
country. Despite regulation, it is widely recognized that the overuse or misuse of antibiotics for the treatments of
zoonotic bacteria is a food safety problem with risk to human health [2]. In addition, the administration route for the
treatment of fish implies the release of large quantities of antibiotics into the environment, resulting in the selection and
spread of genetic determinants of resistance, which affects ecosystems around fish farming operations. In this context,
the research and development of new and safer strategies for controlling bacterial diseases in aquaculture is required.
Because of their biological and chemical diversity, marine environments are sources of alternative products for
prophylaxis and treatment of microbial infectious diseases [3, 4]. Specifically, the great variety of marine molecules
(produced by microorganisms) with surface-active properties known as biosurfactants (BS), offer the potential for
developing innovations for the control of infectious diseases. BS produced by marine bacteria participate in biological
interactions such as intra or inter species communication (quorum sensing) or competition (antimicrobials). They also
have physical properties of industrial interest, such as micelle formation, with potential applications in the development
of functional foods, active coatings (paints), vaccines, and nano structures for bioactive delivery systems.

2. Antibiotics in aquaculture
The infectious diseases caused by microorganisms are affecting aquaculture globally. They attack numerous species
with high incidence, resulting in heavy productive losses. The outbreaks that have occurred in Africa, Asia, Europe and
South America in the last decade have caused partial or total loss of production [2]. For example, Piscirickettsiosis is a
severe condition caused by Piscirickettsia salmonis, which caused losses of US$100 million in only one year of
production in the Chilean salmonid industry [5]. The economic and social impact associated with the main bacterial
pathologies of reared confined fish affect marketable biomass, employment and related business activities.
Unfortunately, the complex techniques of farming and the requirements of commercial production result in raising fish
in high densities that favor stress and, consequently, the emergence and rapid spread of pathogens. In addition, climate

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change and the deterioration of the water quality used for cultivation promote infectious outbreaks [6]. In response, the
aquaculture industry uses large amounts of antimicrobial agents to control microbial diseases. This practice is
considered a risk for aquatic ecosystems, animal and human health, and a food safety problem (Figure 1).

Fig. 1 Impact of antibiotic use in aquaculture.

2.1. Risk for aquatic microbial ecosystems

As mentioned before, the main strategy to prevent and control bacterial outbreaks in fish farms is the administration of
antibiotic therapy on food. Unfortunately, this practice involves the release of antibiotics into aquatic ecosystems since
large amounts of food are not eaten or completely metabolized [7], which has a major environmental impact. Microbial
communities of major importance are affected by the presence and accumulation of these products in sediments and the
water column [8], and microcosm studies show that communities of microorganisms responsible for converting
ammonium to nitrate are highly susceptible to antibiotics such as oxytetracycline [9].
2.2. Risk for animal health
The constant outbreaks caused by different pathogens and the emergence of drug-resistant infectious diseases,
combined with the complex tasks involved in administering antibiotics in aquaculture (incorporated in feed, immersion
or injections) indicate that control measures based on antimicrobial agents are inappropriate [10,11]. Moreover, because
the emergence of drug-resistant bacteria is considered a major issue in aquaculture, the World Health Organization,
together with the FAO and the OIE, are attempting to reduce antibiotic use [2]. Given that antibiotic resistance has been
found in most of the pathogenic species, dealing with these infectious diseases is highly complex [12, 13]. Major
pathogens causing significant pathologies in aquaculture species that require antimicrobial therapy are listed in Table 1.
Table 1 Main pathogenic bacteria in aquaculture.
Pathogen
Aeromonas salmonicida

Pathology
Furunculosis

Listonella anguillarum
(Prev. Vibrio anguillarum)

Vibriosis

Moritella viscosa (Prev.

Vibrio viscosus)
Photobacterium damselae
subsp. piscicida

Winter ulcer

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Photobacteriosis
(Pasteurellosis)

Virulence Factors
Adhesins, aerolysine*,
serine protease*,
haemolysin, lipase,
glycerophospholipid
cholesterol acyl
transferase*, biofilm
formation *.
Flagellum, siderosphore
anguibactin, haemolysin,
cytotoxins, dermatotoxins,
zinc metalloprotease
Vibriolysin, citotoxin
protein
Protease, phospholipase,
lipase, polysaccharide
capsular material,
siderophore-mediated ironsequestering system.

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Main affected species

Atlantic salmon,
rainbow trout,
zebrafish

References
[14,15]

Pacific and Atlantic

salmon, rainbow trout,
turbot, seabass,
seabream, striped bass,
cod, European and
Japan eel, and ayu
Atlantic salmon

[14]

Striped bass, Atlantic

salmon, yellowtail,
ayu, red seabream,
seabass

[17,18]

[16]

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Tenacibaculum maritimum

Flexibacteriosis

Adhesion, biofilm,
exotoxins, proteolytic
compounds, high-affinity
iron-uptake mechanisms.

Pseudomonas
anguilliseptica

Pseumonadiasis

Capsular (K) antigen,

proteinases, lipases

Renibacterium
salmoninarum
Mycobacterium marinum

Bacterial kidney
Disease
Mycobacteriosis

Cytolysin, haemolysin.

Piscirickettsia salmonis

Piscirickettsiosis

Not specified

Erp protein, actin

polymerization,

Turbot, sole, gilthead

seabream, seabass, red
seabream, black
seabream, flounder,
salmonids
Eel, ayu, black
seabream, turbot, black
spot seabream
Salmonids
Pacific and Atlantic
salmon, pejerrey,
snakehead fish, turbot,
tilapia, European
tilapia, red drum.
Pacific salmon,
Atlantic salmon,
rainbow trout, coho
salmon.

[19]

[14]
[20]
[21]

[14]

* Virulence factors under quorum sensing regulation

Chile is the second largest producer of salmon in the world. It is also one of the major users of antibiotics for
aquaculture [22]. Despite the latter, outbreaks of P. salmonis have caused major losses [5], which exemplifies that the
use of antibiotics does not control outbreaks. A major factor affecting efficacy of antibiotic therapy is the oral
administration route for these treatments. Infected fish suffer loss of appetite. For this reason, they do not ingest the
adequate antibiotics doses, and the disease progresses from an asymptomatic condition to evident deterioration and
death. During these stages, infected fish act as vectors in spreading bacterial pathogens. Because of the high-density
conditions, and the inability to identify, separate and confine infected animals, disease progress rapidly. At the same
time, rearing conditions on fish farms favor the evolution of bacterial pathogens to antibiotic resistance.
2.3. Risk for human health and food safe
The antibiotic presence in microbial communities exerts positive selection pressure favoring antibiotic resistant and/or
hypermutator strains to acquire genetic determinants for resistance by mutation or horizontal gene flow [23-25].
Microorganisms positively selected by antibiotics become reservoirs for resistance and when are pathogens cause
antibiotic resistant diseases in plants, animals and humans. While it is true that species barriers limit bacterial
pathogens, the spread of antibiotic resistance appears to be a more promiscuous mechanism that finally favors
microbial evolution. The most serious problem with the antibiotics used to treat infectious diseases in aquaculture is
that they are the same as those employed in human medicine. In fact, according to the WHO, the antibiotics used in
aquaculture (aminopenicillins, macrolides, quinolones, fluoroquinolones and tetracyclines) are critical in human
medicine [26, 27]. Major antibiotics used in aquaculture are listed in Table 2.
Table 2 Antibiotics used in aquaculture
Antibiotic
Florfenicol
Oxytetracycline
dehydrate

Sulfadimethoxine
/ormetoprim

Mechanism
of Action
Inhibition of
protein
synthesis
Inhibition of
protein
synthesis

Route

Dosage

Pathogens

Oral

10 mg/kg of
fish/10 days

A. salmonicida

USA*

Salmonids

Oral

60-80 mg/kg of
fish/10 days

USA*

Salmonids
Lobster
Catfish

Inhibition of
DNA
synthesis

Oral

50 mg/ kg of
fish/day, up to 5
days

A. salmonicida,
A. hydrophila,
A. sobria,
Pseudomonas
spp., Cytophaga
psychrophilia,
Chondrococcus
columnaris,
Yersinia
ruckeri,
Haemophilus
piscium.
Edwardsiella
icttaluri
A. salmonicida

USA*

Salmonids
Catfish

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Approved

Target species

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Ampicillin,
Amoxicillin

Inhibition of
cell-wall
synthesis

Oral

50-80 mg/kg of
fish/10 days

Vibrio spp,
Pasteurella
piscicida,
Mixobacterium
spp

Japan, United
Kingdom**

Yellowtail

Neomycin

Inhibition of
protein
synthesis
Inhibition of
protein
synthesis
Inhibition of
protein
synthesis
Inhibition of
protein
synthesis

Oral

50-80 mg/kg of
fish/10 days

Not mentioned

Indonesia**

Shrimp
Ornamental fish

Immersion

20 mg/liter of
water

Not mentioned

China**

Not mentioned

Oral

50-80 mg/kg of
fish/10 days

Not mentioned

India**

Not mentioned

Oral

50 mg/kg of
fish/10 days

Japan**

Yellowtail
Shrimp

Inhibition of
DNA
synthesis

Oral

200 mg/kg of
fish/10days

China**

Salmonids

12 mg/kg of
fish/10 days

Renibacterium
salmoninarum,
Streptococcus
spp.
Yersinia
ruckeri, A.
salmonicida,
Vibrio spp.
Gram negative
bacteria

Japan, United
Kingdom**

(Yellowtail,
Japan)

12 mg/kg of
fish/10 days

Gram negative
bacteria

Noway,
Japan**

Yellowtail,
Japan)

10 mg/ kg of fish/5
days

Aeromonas
spp., Vibrio spp.

Europe***

Salmonids

Doxycycline
Tetracycline
Erythromycin

Sulfamerazine,
Sulfamethoxine,
Sulfaguanidine

Inhibition of
Oral
DNA
synthesis
Inhibition of
Injected
Flumequine
DNA
synthesis
Oral
Inhibition of
Sarafloxacine
DNA
synthesis
* Approved by the Food and Drug Administration [28].
** Approved by local regulatory agencies [29].
***Approved by the European Medicines Agency [30].
Oxolinic acid

Epidemic aspects of antibiotic resistance have been extensively studied in humans, but clinical evidence connecting
antimicrobial-resistant bacterial infections in persons to the extensive (mis)use of antibiotics in aquaculture remains
under discussion. However, it is clear that the genetic determinants for resistance can be mobilized among different
genera of bacteria by horizontal flows in the aquaculture ecosystem [31-35], the food chain [36,37] and in the human
microbiome [38]. In this scenario, aquaculture ecosystems exposed to antibiotics act as reservoirs for antibiotic-resistant
genes and strains that, by different routes, can affect human health, directly or indirectly (Fig. 1). Direct effects include
the infection of humans by contact with aquatic antibiotic-resistant bacteria, while indirect affects include the
mobilization of bacteria and/or genes with antibiotic resistances through drinking water or foods produced by
aquaculture. Given the above, the World Health Organization declared that the use of antibiotics to control diseases in
aquaculture represents a risk to human health [26]. Considering the increasing quantities of food produced by
aquaculture; food processing (manipulation, storage and delivery) tasks and; eating habits, food consumption can be
regarded as the most important route for the flow of antibiotic resistance to humans. For example, there is a risk in
consuming food produced by aquaculture and contaminated with antibiotic-resistant bacteria if it is not cooked at an
adequate temperature. Unfortunately, this is increasingly common given the wide range of aquacultural food
products and the growing custom of consuming raw fish and shellfish. Thus, efforts should be made not only

to reduce antibiotics in aquaculture, but also to control and diagnose genes and antibiotic-resistant
microorganisms in food produced by aquaculture.

3. Marine bacterial biosurfacants

The highly heterogeneous physical-chemical and biotic conditions in oceans drove marine microbial evolution toward
greater diversity, with a wide array of metabolic and physiological adaptations. Consequently, a great variety of
molecules can be obtained from marine microorganisms. Many of these molecules, by their surface-active properties,
perform their function outside the cell in order to interact with other molecules or other cells. These compounds have
been termed biosurfactants and are amphiphilic molecules or cellular structures being their main characteristic the
affinity for both organic and aqueous phases. The chemical structure of biosurfactants is composed by a variable
hydrophilic moiety (ester or alcohol group of neutral lipids; carboxylate group of fatty acids or aminoacids; phosphate
group of phospholipids; and the carbohydrates of glycolipids) and a more constant hydrophobic moiety (length-variable
fatty acids) (Fig. 2).

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Fig. 2 Biosynthesis and assembly of hydrophilic and hydrophobic moieties of surface-active molecules

In general, hydrophilic and hydrophobic moieties are synthesized and assembled through specific biosynthetic
pathways that, depending on the microorganism and the biosurfactant, involve non-ribosomal peptide synthetases,
glycosyl-transferases, amino-transferases, acyl-transferases and oxidoreductases, which produce a wide variety of
surface-active glycolipids, lipopeptides, glycolipopeptides, phospholipids, acylated serine-lactones and hydroxy fatty
acids (Fig. 2) [39]. As shown in Table 3, marine bacterial biosurfactants can be produced by different genus and species
of microorganisms. They present diverse chemical structures and have wide range of applications, depending on their
physical, chemical and biological properties. Hydrocarbon-degrading marine bacteria (HDMB) are an interesting source
of BS, producing amphiphilic compounds in response to the presence of hydrophobic (aromatic or aliphatic)
hydrocarbons, increasing the bioavailability of these substrates used as carbon and energy sources [40]. Nevertheless,
the production of biosurfactants have been also studied and characterized in other non-HDMB (Table 3).
3.1. Physical-chemical properties
Biosurfactants are a diverse group of compounds with a common structural pattern; a hydrophilic group termed the
hydrophilic head, attached to a hydrophobic group termed the hydrophobic tail, and can be found in monomers or
forming micellar structures. These chemical structures allow biosurfactants to interact simultaneously with hydrophilic
and hydrophobic phases. This phenomenon reduces interfacial and surface tension, in the case of two immiscible phases
(solid-liquid or liquid-liquid) and in the case of a liquid phase and air, respectively. Micelles are physical structures
formed by the aggregation of monomers, generating two separated environments between immiscible phases. At low
concentrations, surfactants form a monolayer in the interface of two these phases. It is only when they reach an
adequate concentration that they aggregate and form micellar structures (Fig. 2). This concentration value is termed a
critical micellar concentration (CMC), and is the lowest concentration at which the monomers of surfactants aggregate
to form micelles. When this phenomenon occurs between two liquid phases and one phase is dispersed into the other, it
is called an emulsion. There are different kinds of emulsions, the most common being: oil-in-water (o/w), where the
hydrophobic phase remains inside the structure surrounded by the aqueous phase; water-in-oil (w/o), where the
hydrophilic phase is inside the micelle and the hydrophobic phase surrounds the structure (Fig. 2). In addition to their
physical and chemical characteristics, the biological properties of biosurfactants make them of interest to the food and
aquaculture industries.
3.2. Biological Properties
In marine ecosystems, amphiphilic surface-active compounds are involved in different biological processes, such as:
microbial competition, when they exhibit antimicrobial properties; cell-to-cell communication, when they act as
diffusible signals in quorum sensing; nutrition, when they favor the accession and assimilation of water-insoluble
nutrients; and survival when they bind and sequester toxic compounds. Because of the variety of roles of bacterial
biosurfactants in nature, they are an interesting alternative to conventional therapies in animal and human health. Given
the great diversity of chemical, biological and physical properties of biosurfactants (Table 3), they represent an
interesting source for a new generation of molecules to prevent and control bacterial infections by inhibiting virulence.

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Bacterial virulence is a set of mechanisms that enables pathogenic cells, accessing, colonize and spread in a host. It is
known that some virulence factors of bacterial fish pathogens, such as Aeromonas salmonicida, Vibrio anguillarum and
Yersinia ruckeri, are regulated by quorum sensing (QS) cell-to-cell communication system. This mechanism is
mediated through the secretion of small signals molecules that allows bacterial cells to act in coordination, increasing
the effect on the host organism. Bacterial pathogenic phenotypes controlled by QS include: swarming, biofilm
formation, sporulation and expression of virulence factors as lytic enzymes, adhesion molecules and toxin production
[41, 42]. For example, in A. salmonicida, production of aerolysin, glycerophospholipid cholesterol acyltransferase and
biofilm formation is under QS control [43]. Therefore inhibiting virulence mechanisms of fish pathogens, by inhibiting
QS, has emerged as an alternative to antibiotic agents in aquaculture [44].
Table 3 Marine bacterial biosurfactants and their biotechnological applications.
Classification

Molecular structure

Name

Bacterial strain

Low Molecular
weight

Lipopeptides

Lipopeptide

Bacillus circulans*
Alcaligenes sp. S-XJ-1

Proline lipid

Alcanivorax dieselolei B-5

Oil-recovery in extreme
Environments

Tyrosine lipid

Alcanivorax
A-11-3T

Alkane degradation

Hydroxy fatty
acids

Cobetia sp. MM1IDA2H-1

Hydroxyl
fatty acids
and iso-fatty
acids

Myroides sp. SM1

Polypeptides

Polypeptide

Acinetobacter sp. A3

Glycolipids

Glucose lipid

Alcaligenes sp.

Bioremediation of crude
oil
Antimicrobial activity

Glucose lipid1

Alcanivorax borkumensis

Oil degradation

Threalose
lipid

Arthrobacter sp.

Oil recovery

Lipoproteins

Ornithine
lipids

Myroides sp. SM1

Oil remediation

[55]

Lipopolysaccharides

BD4 Emulsan

Acinetobacter calcoaceticus
BD4

oil/water emulsion
stabilization

[56]

Emulsan2

A. calcoaceticus RAG-1

Stabilization of oil/water
emulsions

Biodispersan

A. calcoaceticus A2

Limestone powder
dispersion

Exoplysaccha
ride

Halomonas eurihalina*

Oil pollution
bioremediation

HE39
HE67

Halomonas sp .TG39*
Halomonas sp.TG67*

Bioremediation

Yansan

Yarrowia lipolytica IMUFRJ

50682*

Polymeric BS

Pseudomonas nautica 617

Fatty Acids

High
molecular
weight

Polysaccharide

Glycoproteins

Polymeric BS

hongdengensis

Biofilm inhibition**
Nanostructures
formation
Inverse micelles
formation
Inhibition of virulence
factors in fish
pathogens**

Reference
[45-48]

[49,50]

Oil recovery

* Non hydrocarbon-degrading bacteria.

** Inhibition of the Quorum Sensing communication system.

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Biotechnological
properties
Antimicrobial activity
Demulsifier

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Bioremediation
Bioremediation /
Formulation of
perfluorocarbon-based
emulsions
Bioremediation

[51]
[52-54]

[57,58]

[59]
[60,61]

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3.3. Antimicrobial activity and quorum sensing inhibition

As presented in Table 3, biosurfactants of marine origin have diverse structures, properties and potential applications.
Among them, lipopeptide of Bacillus circulans and the glucose-lipid of Alcaligenes sp. have been demonstrated to have
antibacterial properties, but their mechanisms of action have not been specified. Other marine biosurfactants of interest
for the prophylaxis and control of bacterial infections are those compounds with fatty acid structure. It has been
reported that fatty acids from biological sources can inhibit the quorum sensing communication system in human
pathogenic bacteria such as Proteus mirabilis and Escherichia coli [62,63] In this context, hydroxyl fatty acids from
Cobetia sp. MM1IDA2H-1 offer an alternative way to control infectious outbreaks in fish farming, due to their ability
to disrupt the QS communication system of fish pathogenic bacteria such as A. salmonicida and L. anguillarum [49].
This fatty acid inhibit the expression of virulence factors such as enzyme synthesis and biofilm formation, by hijacking
the signal molecules involved in QS. This mechanism does not involve interaction with cellular structures and,
therefore, no resistance is developed. QS can be inhibited at different levels, for example, compounds that antagonize
the receptor of the signaling molecules can also inhibit cell-to-cell communication and alter bacterial pathogenicity.
Another mechanism that interrupts QS is inhibiting signaling molecule synthesis. Molecules with structural analogy to
chemical substrates need to form acyl-homoserine lactones, which can be inhibited. In this context, the great variety of
chemical structures of biosurfactants produced by marine bacteria offer an interesting source for compounds that inhibit
quorum-sensing systems.
3.4. Immunomodulation
In addition to inhibiting growth, dissemination and virulence of pathogenic bacteria, to prevent infectious outbreaks it
important to consider it the efficiency of the fish immune system of fish to confront and control pathologies [64]. The
main immunological response in fish is based on the innate immune system [65,66], which can be activated by several
molecules, such as lipopolysaccharides, lipoproteins and glycoproteins of bacterial origin, as well as by enzymes
produced by immune cells, such as cytokines, transferrin, lysozyme and interleukins [67,68]. The innate immune
system of fish involves receptors responsible for activating immune and pro-inflammatory responses called toll-like
receptors or TLRs [67,68]. It has been reported that biosurfactants can act as immunostimulating molecules [69,70],
making marine bacteria an interesting source of compounds to strengthen fish immune system and, thus, reduce the
quantity of antibiotics required to control infectious outbreaks.

4. Innovations based on marine microbial biosurfactants

Although synthetic surfactants are currently the most commonly used surface-active compounds, the main advantages
of biosurfactants are their low toxicity, high biodegradability and high stability at extreme conditions, such as high
temperatures and high levels of salinity and pH [71, 72]. In some cases, stability is higher than that of chemically based
surfactants [73]. Biosurfactant molecules can form stable emulsions in solutions with high ionic force, which is
especially relevant in aquacultural applications, since most fish farms are located in high salinity environments, making
the stability of biosurfactants at high ionic strength essential. For example, biosurfactants from Cobetia sp.
MM1IDA2H-1 [49] and emulsions formed from this compound present high stability at wide ranges of pH, temperature
and strength force (3-20 % w/v of sodium chloride).
Synthetic surface-active compounds have been classically used in the food industry as food additives and
emulsifying agents. Due to the emulsifying properties and high stability of emulsions the marine bacterial bioactive
biosurfactants can be incorporated into food. For example, nowadays, the incorporation of antibiotics in the fish feed is
accomplished during the extrusion of the feed. In this case due to the ability to form stable emulsions, biosurfactants
with remarkable biological functions can be incorporated (after the extrusion process) during the food oiled. In this
context, biosurfactants can be incorporated into polymeric food matrices by emulsifying the food components.
Likewise, because of the ability to form micelles, biosurfactants can be used as a delivery system for a variety of
bioactive molecules in order to improve the fish health (Fig. 2). This allows the incorporation of compounds into
aqueous and organic phases during feed manufacture.
The main application of biosurfactants has been forming oil and water emulsions with applications in bioremediation
and the food industry (Table 3). Nevertheless, water-in-oil micelles, also called reverse micelles, have applications in a
several fields. The core structure of reverse micelles is formed by an aqueous phase dispersed in an organic phase.
Therefore, these structures have a major potential for the recovery, separation and purification of bioactive hydrophilic
molecules [74-78]. Because of their chemical structure and amphiphilic nature, reverse micelles also have potential
application for administering hydrophilic drugs since they can pass through biological membranes and release active
molecules [79]. Reverse micelles can also reduce the degradation of oxygen sensitive molecules such as citral, with
potential applications in food, beverage and perfume production [80]. A remarkable application of reverse micelles is
their ability to host hrydrophilic enzymes in the water core, and hydrophilic enzymes with hydrophobic substrates are of
particular interest [81-83]. Water-in-oil emulsions can also act as a delivery system for inorganic antimicrobials like
silver nanoparticles [84]. Although the majority of reverse micelle applications involve synthetic surfactants, marine

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biosurfactants offer an interesting alternative. For instance, by the ability to form stable emulsions in different chemical
conditions, is possible to use reverse micelle technology to manufacture bioactive polymer coatings for preventing
biofilm formation on surfaces submerged in the aquaculture installations. Biofilm formation in farm facilities is a
reservoir of potential microbial pathogens. Biofilm formation in the facilities of the farm is a reservoir of potential
microbial pathogens. Therefore, paints containing heavy metals contaminants are used to prevent biofilm formation.
Then, using paints without contaminants to prevent biofilms with active environmentally friendly innovations is a
technological breakthrough.
Acknowledgements The support by grant InnovaChile 12IDL4-16167 by Corfo, Chilean government is gratefully acknowledged.
C. Ibacache is supported by BecasChile fellowship program from the Chilean Government.

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