Biological Control 67 (2013) 380–389

Contents lists available at ScienceDirect

Biological Control
journal homepage: www.elsevier.com/locate/ybcon

Review

Review of encapsulation methods suitable for microbial biological

control agents
Marina Vemmer, Anant V. Patel ⇑
University of Applied Sciences, Wilhelm-Bertelsmann-Str. 10, 33602 Bielefeld, Germany

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 We present a detailed overview on

materials, capsule morphologies and
methods.
 We particularly refer to the suitability
for biological control agents.
 We put emphasis on chemical
aspects.

a r t i c l e i n f o a b s t r a c t

Article history: Because of the rising demand for microbial biological control agents, research into novel formulation
Received 15 October 2012 methods, especially bioencapsulation, has notably increased in the past years. The aim of this review
Accepted 3 September 2013 is to present a detailed illustrated overview on current encapsulation methods that are applied or that
Available online 12 September 2013
may be tailored to living biological control agents, especially microbial organisms and entomopathogenic
nematodes. Capsules are manufactured by forming droplets from liquids and solidifying the liquid drop-
Keywords: lets to form particles. In this review, the methods are presented according to the manner of droplet for-
Formulation
mation (dripping and emulsification) and are subsequently categorized by the process of gelation or
Encapsulation
Biological control
membrane formation. In a further category this review expands on coating methods using polyelectro-
Biopesticide lytes with altering charges. We put emphasis on chemical aspects which seem especially useful for sci-
Microbial pesticide entists working in biological control.
Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction (micro)encapsulation technologies in the major industrial areas

It is well established that encapsulation methods are suitable
for formulation of a wide range of active ingredients starting from
the microencapsulation of inks in the 1950s (Green, 1955) to
highly innovative capsule systems for pharmaceutical applications
(Del Valle, 2004; Hunt and Grover, 2010; Orive et al., 2004;
Vidhyalakshmi et al., 2009) and food technology (Gibbs et al.,
1999; Groboillot et al., 1994; Onwulata, 2012; Shahidi and Han,
1993) to name a few. In its report, the market research company
Frost & Sullivan (2002) gives an overview of the use of
⇑ Corresponding author. Fax: +49 521 106 7152.
E-mail address: anant.patel@fh-bielefeld.de (A.V. Patel).
in Europe. The number of encapsulation-related publications is
increasing rapidly. In recent years there were almost 2500 publica-
tions per year whereof over 1500 are patents (Poncelet and Boh,
2008). Consequently, a great variety of encapsulation methods is
available for the formulation of biological control agents, especially
microbial agents and entomopathogenic nematodes, on which we
will focus in this review. We will use the term ‘‘microbial pest con-
trol’’ when microorganisms are used for pest control purposes and
we expand this term on entomopathogenic nematodes, according
to Ravensberg (2011), although they are not generally defined as
microorganisms. Biochemical or natural pesticides based on non-
living structures such as pheromones (Leonhardt, 1990) or plant
extracts (Isman, 2000; Moretti et al., 2002; Varona et al., 2009)

1049-9644/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.biocontrol.2013.09.003
 M. Vemmer, A.V. Patel / Biological Control 67 (2013) 380–389
are beyond the scope of this review. The pest control disciplines
concerned are the biocontrol of plant pathogens, pest insects and
plant pathogenic nematodes.
Since microbial control includes the use of sensitive living
organisms a suitable encapsulation system has the potential to im-
prove the characteristics of a microbial biological control agent
with regard to an extended shelf life, a decreased number of appli-
cations and a reduced dose, a reduced biomass content as well as
an improved handling (Bashan, 1998, 1986; Cassidy et al., 1996;
McLoughlin, 1994; Mnyone et al., 2009; Vassilev et al., 2001a,b,c).
As depicted in Fig. 1, encapsulation within a matrix protects the
microbial biological control agent from biotic and abiotic stress
factors (contaminations, soil antagonists, temperature, dryness,
UV light, mechanical stress) by providing a beneficial microenvi-
ronment. This leads to an extended shelf life and to the mainte-
nance of the metabolic activity for extended periods of time not
only during storage but also after application resulting in a
decreased number of applications, a reduced dose as well as a re-
duced biomass content. The maintenance of the metabolic activity
may be further improved by providing nutrients which allow the
construction of ‘‘mini-fermenters’’ with extremely low initial bio-
mass content thus saving on biomass and making formulations
much more cost-effective. Besides, as a function of the material
properties the cells may be released slowly by growth out of the
matrix or degradation of the encapsulation material, leading to
an increased establishment in soil or on the leaf and an extended
persistence after application, resulting again in a decreased num-
ber of applications and a reduced dose. A further option to improve
the characteristics of a microbial biological control agent is the
co-encapsulation of other microorganisms or non-living active
ingredients such as semiochemicals and chemical pesticides,
which often interact synergistically at reduced dose (Paula et al.,
2011; Wang et al., 2002). The targeted delivery of a biological con-
trol agent at a certain point of time at a certain location to a certain
insect is an important feature which decreases the number of
applications and reduces the dose. Especially for co-encapsulated
low-dose chemicals the environmental impact is reduced. Targeted
delivery can be achieved by controlled release systems triggered by
environmental conditions and material properties as well as by
Fig. 1. Advantages of encapsulated microbial biological control agents.
381
novel co-formulations following an ‘‘attract and kill’’ strategy
(Vemmer and Patel, 2011).
Furthermore, encapsulation allows the isolation of the active
ingredients during the application leading to an improved protec-
tion for workers, e.g. from allergenic spores or co-encapsulated
pesticides. This reduced dusting and the generally improved char-
acteristics that arise from the formation of solid formulations like
dosability, mechanical stability and flowability, result in an im-
proved handling.
Despite this high potential, few encapsulation approaches are
investigated for applications in biological control. Nedović and
Willaert (2005) give a broad overview on applications of cell
immobilization biotechnology including microbial cells. Environ-
mental applications of immobilized microbial cells are reviewed
by Cassidy et al. (1996). Burges (1998) approaches the formulation
of microbial biopesticides including nematodes, too. However,
there are still few systematic studies on encapsulation materials
and methods for microbial biological control agents.
The aim of this review is to present an extensive overview on
current encapsulation methods that have already been investi-
gated or that may be tailored to living microbial biological control
agents. We put emphasis on chemical aspects which seem espe-
cially useful for scientists working in biological control.
2. Materials
The polymers that are used to form the basic capsule matrix are
commonly referred to as ‘‘capsule materials’’. It is often not under-
stood that the resulting capsules are made of more than just the
polymer such as counter ions, residues of acids or bases to adjust
pH values, residues of solvents as well as water. Besides, capsules
will contain 1–5 synergistic additives to further improve product
properties. Thus, capsule material characteristics do not automati-
cally imply capsule characteristics.
Suitable materials for capsule construction are biodegradable
polymers (Chandra and Rustgi, 1998; Endres and Siebert-Raths,
2009). These are, more specifically, natural polysaccharides like
alginates, carrageenans, agar/agarose, gellan gum, guar gum, acacia
gum, starch, starch-based materials, cellulose, pectin, chitosan
382 M. Vemmer, A.V. Patel / Biological Control 67 (2013) 380–389

Table 1
Most important characteristics of materials for biological control.

Physico-chemical Molecular weight and distribution

properties
Economic factors
Safety and quality
Degree of substitution
Counter ions
Gelation mechanism
pH
Viscoelastic properties (rheology)
Primary, secondary and tertiary structures
Costs (roughly 10–100 €/kg; depending on
application)
Toxicity
Degradability
FDA or EPA approval except nematodes
Source, batch no.

including derivates thereof (Grasdalen and Smidsrød, 1987; Guise-
ley, 1989; Murano, 1998; Pérez and Mazeau, 2005; Smidsrød and
Skjårk-Bræk, 1990; Wandrey et al., 2010; Whistler, 1993; Yi
et al., 2005), polypeptides such as poly-L-lysin (Obst and Steinbü-
chel, 2004) or proteins like gelatin or whey (Fakirov, 2007; Wan-
drey et al., 2010), lipids like waxes (Wandrey et al., 2010) or
other biopolymers like lignin (Endres and Siebert-Raths, 2009;
Hatakeyama and Hatakeyama, 2010). Besides, a wide variety of
classic synthetic polymers like polyurea, polyurethanes, polya-
mides, polyesters polyvinylalcohol, polyacrylates, silica-based
polymers as well as novel co-polymers (Endres and Siebert-Raths,
2009; Kirk-Othmer Encyclopedia of Chemical Technology, 2000;
Peteu et al., 2010) are available. All these materials differ in certain
characteristics (Table 1) which is of utmost importance for the
application of encapsulation methods to microbial biological con-
trol agents.
3. Capsule morphology
There is confusion in scientific literature about encapsulation
shapes and structures because of different definitions. Depending
on the encapsulation method, capsules may assume different
morphologies (Fig. 2). The most common shape of capsule is that
of a spherical design. This design may be subdivided into different
types of structures. The term ‘‘beads’’ defines solid spheres
whereas the term ‘‘hollow beads’’ defines capsules with a liquid
core. In classic biotechnology, the latter capsule system where
the biocatalyst is retained behind a semipermeable membrane is
defined as ‘‘microcapsule’’, independently of the size. However,
some authors define microencapsulation as production of capsules
in microsize (10–100 lm Ø) as opposed to macroencapsulation
(>100 lm Ø) (John et al., 2011; Lin and Chen, 2002). Beads and hol-
low beads may be surrounded with appropriate materials to form
‘‘coated beads’’. Clever combinations of structures will allow the
design of ‘‘multicompartment beads’’ with multiple cores and/or
coatings. Droplets may also be extruded onto a flat surface result-
ing in lens-like shape capsules. In all these capsule systems, one or
several microbial biological control agents and additives may be
incorporated.
4. Methods
Usually, capsules are manufactured by forming droplets from
liquids by either dripping or emulsification and solidifying the
liquid droplets to form particles. The following illustrates encapsu-
lation methods by droplet formation (dripping and emulsification)
and ‘‘solidifying principle’’ (gelation mechanisms or membrane
formation at the droplet surface). The methods are subdivided into
the manner of droplet formation and are subsequently categorized
Fig. 2. Different capsule morphologies: beads, hollow beads, lens shape particles,
coated beads and multicompartment beads.
by the process of gelation or membrane formation. As a further
category we expand on coating of capsules which can be applied
in many cases to improve capsule properties.
Especially for biocatalysts (enzymes and whole cells), a lot of
literature on immobilization and encapsulation is available (e.g.,
Buchholz et al., 2005; Campàs and Marty, 2006; Sheldon, 2007).
Renken and Hunkeler (1998) reviewed polymers and technologies
suitable for microencapsulation with a focus on bioartificial
organs. Dulieu et al. (1999) give an overview on encapsulation
and immobilization techniques regarding cell encapsulation
technology and therapeutics. The manual ‘‘Immobilized Cells’’
presents simple laboratory methods for the immobilization of cells
(Wijffels, 2001).
Only some of the encapsulation methods suitable for living cells
are currently applied to microbial biological control agents or
entomopathogenic nematodes. Most encapsulation methods de-
scribed for applications in microbial pest control are based on ionic
gelation using alginate (Connick, 1988; McLoughlin, 1994). Never-
theless, other methods that are not yet applied in microbial pest
control but that are dealing with the encapsulation of living cells
may be tailored to the needs of a microbial agent. Bioencapsulation
of microbial cells used as biofertilizers or biocontrol agents for tar-
geted agricultural delivery has been reviewed recently (John et al.,
2011). However, a detailed overview emphasizing chemical
aspects of all current encapsulation methods with potential for
applications in microbial pest control has been missing in litera-
ture so far.
4.1. Dripping
A simple method to form droplets is to extrude a liquid through
a nozzle and allow the droplet to harden e.g., in a suitable solution.
The simplest approach is using a syringe connected to a needle.
This method shows a low particle size distribution, but, depending
on the diameter of the needle, it results in relatively large beads
(1–4 mm) and the production capacity is limited by the speed of
droplet formation. Dripping devices with pressure applications,
an increased number of needles or droplet breaking systems (coax-
ial air flow, electrostatic droplet generator, nozzle resonance meth-
od, and jet-cutting method) are used to obtain smaller droplets at
larger flow rates. A comparative overview of common capsule
production technologies based on dripping (in the case of alginate)
is given in Prüsse et al. (2008). Commonly used encapsulation
techniques based on dripping and jet break-up for medical and
biotechnological applications have recently been compared
 M. Vemmer, A.V. Patel / Biological Control 67 (2013) 380–389 383

(Whelehan, 2011). The following explains dripping methods suit- Gelatin, agar and agarose form thermally reversible gels that do
able for microbial control agents (Fig. 3). not require ions for gel stabilization. A simple method for the
encapsulation of cells within gelatin is to harden a gelled block
containing the living cells by crosslinking agents and dissect it into
4.1.1. Thermal gelation
pieces (De Alteriis et al., 1990; Dhulster et al., 1983; Gianfreda
Some polymers such as carrageenan, gellan gum, gelatin, agar
et al., 1980). This method was transformed into dripping methods
or agarose gel by a temperature decrease. The gelation is thermally
resulting in spherical beads (Prüsse et al., 2000; Willaert and Bar-
reversible so that gels may soften or disintegrate at elevated
on, 1996).
temperatures. Beads may be obtained by dripping such a warm
polymer solution containing the living cells into a cold solidifying
4.1.2. Ionic gelation
solution (Fig. 3A).
4.1.2.1. Beads. About 50 years ago, Thiele and Andersen (1955),
In the case of j-carrageenan kalium ions will aid in the gelling
Thiele and Cordes (1967) observed the formation of a highly or-
process by stabilizing the gel structure. The immobilization of
dered hydrogel when a solution of divalent cations is brought into
microbial cells using carrageenan is a common method (Takata
contact with a solution of some polyuronic acids such as alginates.
et al., 1977; Tosa et al., 1979).
For the formation of beads a solution of sodium alginate, sodium
Gellan gelation is induced by cooling in the presence of divalent
pectinate or guar gum derivates containing the living microbial
cations (Grasdalen and Smidsrød, 1987). As encapsulation material
biological control agents is dropped into a crosslinking solution
for whole cells gellan gum was first used for the immobilization of
containing divalent cations, e.g., Ca2+. Solidification of droplets
thermophilic organisms (Norton and Lacroix, 1990). For the
starts within milliseconds on the droplet surface by ionotropic
entrapment of mesophilic bacteria the gelation temperature may
gelation where Ca2+ ions react with the negatively charged
be decreased by addition of chelating agents (Camelin et al., 1993).

Fig. 3. Encapsulation methods based on dripping: Thermal gelation (A), production of beads (B) and hollow beads (C) obtained by ionic gelation, spray drying (D), complex
coacervation (E), Production of LentikatsÒ (F) and sol–gel immobilization based on silicon chemistry (G).
384 M. Vemmer, A.V. Patel / Biological Control 67 (2013) 380–389
polymer chains to form a three-dimensional rigid structure con-
taining water, a hydrogel. Through this water Ca2+ ions will diffuse
into the liquid droplet thus crosslinking ‘‘from outside to inside’’
(Fig. 3B). This gelation method for immobilizing cells, subcellular
organelles and enzymes is very well studied in the case of alginates
(Hackel et al., 1975; Kierstan and Bucke, 1977; Klein and Vorlop,
1983; Lim and Sun, 1980; Martinsen et al., 1992, 1989). Alginate
is the most widely used material for the formulation of microbial
biopesticides. As reviewed already by Connick (1988), conven-
tional calcium alginate beads were used for the incorporation
and delivery of several fungi (Alternaria, Fusarium, Phyllosticta
spp., Trichoderma, Gliocladium, Talaromyces and Penicillium spp.)
and bacteria (Pseudomonas and Bacillus spp.), which can control
soilborne plant disease pathogens, and insect-killing nematodes
(Steinernema and Heterorhabditis spp.).
Variations of this method use pectinates and derivates, guar
gum derivates and sometimes calcium gluconate instead of cal-
cium chloride or even other divalent metallic cations like Ba2+
and Cu2+ ions (Prabakaran and Hoti, 2008).
4.1.2.2. Hollow beads. With the same solutions as above, hollow
beads may be obtained by just switching the positions (Spieker-
mann et al., 1987): A crosslinking solution is supplemented with
a viscosity-enhancing polymer such as sodium carboxymethylcel-
lulose that does not participate in the reaction and then dripped
into an alginate solution. Again, at the surface ionotropic gelation
occurs, but this time, calcium ions will diffuse from the core to
the surface crosslinking ‘‘from inside to outside’’ (Fig. 3C). The
thickness of the calcium alginate layer around the core may be
controlled by the concentrations of the reactants and the crosslink-
ing time. Cells may be placed either into the core and/or into the
alginate layer. The core can also become solid if a warm gelatin
or agar solution is used instead of carboxymethylcellulose. Regard-
ing biological control, this method was applied to the encapsula-
tion of an entomopathogenic nematode into calcium alginate
hollow beads (Patel and Vorlop, 1994).
A methodological alternative for the production of hollow beads
for the encapsulation of bacteria, fungi, nematodes and viruses is
the use of coextrusion where a core solution is extruded into a sur-
rounding shell solution (Digat, 1993).
4.1.3. Spray drying
While spray-drying is not considered a classic encapsulation
method, there are still publications claiming the term ‘‘encapsula-
tion’’. Briefly, a polymer solution is dropped (dispersed) into a hot
air stream. The solvent (water) evaporates on the way to the bot-
tom leaving a dried particle (Fig. 3D). The main advantage is the
combination of particle formation and drying in one step. The dis-
advantages from the viewpoint of biological control are the varia-
tions in particle shape and size distribution, high temperatures and
fast drying rates that normally do not allow for encapsulation of
drying-sensitive living cells such as non-spore forming microbials.
Protectants and other additives may be added before or during the
drying process. Especially in the food industry, spray-drying is
used for the preservation and concentration of microorganisms
(Knorr, 1998; Meng et al., 2008; Menshutina et al., 2010). In biolog-
ical control, too, spray drying is used to produce formulations with
extended shelf-life (McGuire et al., 1996; Muñoz-Celaya et al.,
2012; Tamez-Guerra et al., 1996).
4.1.4. Complex coacervation (polyelectrolyte–polyelectrolyte complex
formation, symplex gel formation)
Complex coacervation is the reaction of two dispersed water-
soluble polymers of opposite electric charges forming a dense
coacervate phase and a dilute equilibrium phase. It was discovered
in 1930 (Bungenberg de Jong and Kruyt, 1930) and was already
well-investigated in the 50s (Overbeek and Voorn, 1957). One of
the oldest technical applications is located in the printing industry
where gelatin/gum arabicum capsules containing ink were used for
the development of pressure sensitive copy paper (Green, 1955).
More precisely, complex coacervation can be explained as
follows: Polyelectrolytes are macromolecules carrying multiple
functional charged or chargeable groups. Because of the dissocia-
tion of acid or basic groups some molecules possess charges when
dissolved in water. This leads to repulsion between identically
charged molecules and to an increased hydration compared to
uncharged molecules. When two oppositely charged colloids
dissolved in water are combined, they will be attracted by each
other compensating their charges. This results in a reduced hydra-
tion and in the formation of small droplets consisting of a slightly
hydrated complex colloid (coacervate phase). This process is af-
fected by pH, temperature, ionic strength, molecular weight and
concentration.
For the production of cell containing hollow beads, the complex
coacervation method encompasses dripping a solution of a poly-
electrolyte, e.g., a polyanion such as cellulose sulfate containing
the living cells into a solution of another polyelectrolyte with
counter charges, e.g., a polycation such as polydiallyl dimethyl
ammonium chloride (PDADMAC). At the droplet surface, an ionic
reaction between the two polyelectrolytes occurs (Dautzenberg
et al., 1985, 1996) forming a thin semipermeable membrane
around a liquid core (Fig. 3E). Variations include other polyanions
such as sulfoethyl cellulose and other polycations such as chitosan
(Patel, 1998; Rose et al., 2000). In such hollow beads, the nemato-
phagous fungus Hirsutella rhossiliensis was co-encapsulated with
nutrients (Patel et al., 2011). A number of other polyelectrolytes
suitable for microencapsulation using complex coacervation was
investigated (Prokop et al., 1998a,b).
4.1.5. LentiKatsÒ
By repeated freeze–thawing polyvinyl alcohol (PVAL) normally
forms hydrogels, so-called cryogels (Lozinsky et al., 1986) which
are incompatible for most living cells (Jekel et al., 1998a). However,
a variation where the gelation of a PVAL-hydrogel occurs at room
temperature was developed. Mixing PVAL with living cells and
glycerol and then extruding it onto a surface will produce lens-
shaped droplets that are then air-dried to a certain water content
(LentiKatsÒ) (Jekel et al., 1998a, 1998b) (Fig. 3F). The resulting
particles are then rehydrated to yield a hydrogel stable at room
temperature with superior diffusion characteristics. Due to its thin
lens-shape internal diffusional limitations are minimized com-
pared to beads resulting in better diffusion of substrates and prod-
ucts into and out of the gel matrix. In contrast to biopolymer
hydrogels LentiKatsÒ also show a low biological degradability
and a high mechanical stability.
4.1.6. Sol–gel immobilization based on silicon chemistry
Biopolymers offer the advantage of biocompatibility but are
considered chemically and mechanically instable (Rooke et al.,
2008). To combine inorganic matrix materials such as glass-like
materials with biomolecules (proteins and enzymes, antibodies,
DNA, phospholipids, polysaccharides) or whole cells the sol–gel
method was developed over the past two decades (Avnir et al.,
2006). Conventional synthesis of silica gels offers the advantage
of chemically, mechanically, thermally and biologically stable
matrices (Livage et al., 2001). However, silica gels and ceramics
produced by sol–gel methods are not biocompatible due to ex-
treme pH, temperature and toxic by-products (Livage and Coradin,
2006). Nevertheless, the potential of inorganic matrices for biolog-
icals such as enzymes was demonstrated: Tetramethoxysilane
(TMOS) and/or methyltrimethoxysilane (MTMOS) are incubated
4 °C overnight in the presence of water and acid pH to allow for
 M. Vemmer, A.V. Patel / Biological Control 67 (2013) 380–389
hydrolysis of the precursor(s) resulting in the formation of silanol
groups (Si-OH). At basic pH, the condensation reaction between
silanol moieties results in the formation of siloxane (Si–O–Si) poly-
mers creating a matrix for the entrapment of biomolecules
(Campàs and Marty, 2006) (Fig. 3G). As an alternative to TMOS
and TEOS, silicic acid was used for entrapment of living cells but
high NaCl concentrations were formed (Meunier et al., 2010).
Other novel silica gels and glass-like materials with more biocom-
patibility based on highly reactive precursors are currently being
developed (Müller et al., 2013).
4.2. Emulsification
As another method to form droplets emulsification provides
smaller particles but with a higher size distribution compared to
dripping methods. These methods are based on the emulsification
of a core material in a non-miscible phase and can be realized as
batch using cylindrical vessels with an impeller (turbine, marine-
style impeller or grid device) or, at large scale, in a continuous
mode using a static mixer, which consists of a series of stationary
elements mounted lengthwise in a pipe (Poncelet and Neufeld,
1996; Poncelet et al., 2001a,b). Emulsification methods suitable
for microbial biological control agents (Fig. 4) are presented below.
4.2.1. Thermal gelation
This method involves emulsifying an aqueous solution of j-car-
rageenan, agarose or agar and cells in warm oil and then decreas-
ing the temperature to induce thermal gelation (Audet and Lacroix,
1989; Nilsson et al., 1983; Raymond et al., 2004) (Fig. 4A). In the
case of j-carrageenan, further stabilization can be obtained with
K+ ions.
Another example for a method using emulsification followed by
thermal gelation is the production of gelatin beads (Nilsson and
Mosbach, 1980; Tanaka et al., 1963).
4.2.2. Ionic gelation
Ionotropic gelation may be used to solidify droplets from emul-
sions. The cell/alginate droplets are gelled following addition of
CaCl2 solution (Nilsson et al., 1983) or by internal gelation (Poncelet
Fig. 4. Encapsulation methods based on emulsification: Thermal gelation (A), ionic gelation (B) and interfacial emulsion polymerization (C).
385
et al., 1995; Wan et al., 1992). The latter method includes the emul-
sification of a mix of sodium alginate, living cells and an insoluble
calcium salt such as calcium carbonate in an oil phase. Adding a
solution of acetic acid in oil will result in the acid lowering the
pH of the aqueous droplets and thus releasing calcium ions from
the salt. These will then crosslink the polymer by internal gelation
towards an ionotropic hydrogel as described in 5.1.2 (Fig. 4B).
Moslemy et al. (2002) developed a method for the encapsula-
tion of a mixed culture of gasoline-degrading bacteria in ion-sensi-
tive, and also thermotropic, gellan gum, which might be easily
transferred to applications in microbial biological pest control.
4.2.3. Interfacial emulsion polymerization
This classic method for the encapsulation of pesticides and
pheromones (Scher, 1999; Wilkins, 1990), involves, for cell encap-
sulation, emulsifying living cells and a hydrophilic monomer A in
an organic phase amended with an emulsifier. By adding a hydro-
phobic monomer B and a catalyst the two building blocks will react
in a polymerization reaction as is used in polymer chemistry (e.g.
synthesis of polyurea, polyurethanes, polyamides and polyesters)
forming a wall at the surface of the droplets (Fig. 4C). Many varia-
tions of this method exist. The main problem for biological control
is the toxicity of the monomers and sometimes the reaction condi-
tions. A solution may be the use of less toxic pre-polymers (Vorlop
et al., 1992).
4.3. Coating
Two useful methods to coat biopolymers onto capsules are ionic
polymer coating and layer-by-layer coating, which both use poly-
electrolytes with altering charges (Fig. 5). Whereas ionic polymer
coating is often connected to cell encapsulation, layer-by-layer
coating originates historically from nanotechnology.
4.3.1. Ionic polymer coating
Coating can be achieved by suspending polyanionic beads (core)
in a polycationic solution (shell). Multicoating is performed by
alternate coatings with polyanionic and polycationic polymers.
A simple example is the coating of calcium alginate beads with
386 M. Vemmer, A.V. Patel / Biological Control 67 (2013) 380–389
chitosan by complex formation between amine groups of chitosan
and carboxyl groups of alginate when alginate beads are trans-
ferred into a 0.2% chitosan solution (Fig. 5A). This first coating
may be followed by a second coating with alginate solution, and
so on (Poncelet et al., 2001c).
Another well-known bead is a calcium alginate bead coated
with a solution of an oppositely charged polypeptide such as
poly-L-lysine (Lim and Sun, 1980; Sun, 1997). Carboxyl groups
of the calcium alginate beads react with amine groups of
poly-L-lysine resulting in the formation of a polyelectrolyte
complex membrane on the surface of the calcium alginate beads.
The solid calcium alginate core may be dissolved by sodium
citrate to obtain hollow beads.
Another coating method makes use of ionotropic gelation (see
4.1.2): When calcium alginate beads are dripped into a sodium
alginate solution, free Ca2+-ions will result in a calcium alginate
bead coating by crosslinking from ‘‘inside to outside’’ (Klein
et al., 1986; Vorlop et al., 1987) (Fig. 5B).
4.3.2. Layer-by-layer coating
A similar way to form multilayer capsules is the layer-by-layer
(LbL) assembly by colloid-templated consecutive polyelectrolyte
adsorption which is mainly used in nanotechnology. This method
involves a stepwise film formation by repeated exposure of col-
loids (templating cores) to polyelectrolytes of alternating charge
(layers). To obtain hollow polyelectrolyte shells the core can be re-
moved when the desired number of polyelectrolyte layers is
reached (Donath et al., 1998) (Fig. 5C). A variation of this method
uses biocrystals as templates for the deposition of polymer multi-
layers (Caruso et al., 2000). The LbL method is well-investigated,
but mostly for nanocapsules (Decher and Schlenoff, 2012; Donath
et al., 2006) which are unsuitable for the incorporation of microbial
biological control agents due to their size. As shown in the field of
tissue engineering living cells can be used as functional elements
of polyelectrolyte multilayers which includes the incorporation of
cells into multilayers and the attachment of multilayers to the sur-
faces of cells (Rubner and Cohen, 2012).
Fig. 5. Coating methods: Ionic polymer coating using Chitosan (A) or Na-alginate (B) on Ca-alginate and layer-by-layer coating (C).
Table 2
Characterization of capsules.
Morphology Size (micro, nano), particle size distribution
Shape (spherical, lentic, undefined)
Structure (beads, hollow-beads, coated beads,
multicompartment systems)
Physico-chemical Diffusional characteristics (cut-off, internal and
properties external diffusion limitations)
Surface charge
Hydrophobicity
Redisolvability by pH, temperature, ion exchange,
enzymatic reactions, reswelling
Mechanical properties Stability
Biological properties Biocompatibility
Biological degradability in soil, persistence
5. Capsule additives
The same argumentation as for the versatility of encapsulation
materials and methods can be made for the manifold additives
where few systematic studies are available. While there are listings
of additives (Bernhard et al., 1998) there are few reliable scientific
data on their action. Modern formulation science involves investi-
gations of the complex, mostly physicochemical interactions of
more than two, often five and more formulation additives that syn-
ergistically interact to improve characteristics (see Chapter 6 and
Table 2) of the product. There are no systematic physicochemical
and biological investigations published on how these additives
interact with each other, with living cells and with the environ-
ment. Most studies remain purely empirical.
6. Characteristics of capsules
To conduct systematic scientific bioencapsulation research the
capsules themselves must be characterized properly (Table 2).
For example, capsule morphology as described in chaper 3
(beads, hollow beads, coated beads, multicompartment systems)
 M. Vemmer, A.V. Patel / Biological Control 67 (2013) 380–389
plays an important role because highly sophisticated structures
can be fabricated that e.g., allow co-encapsulation of several agents
or one agent with a synergistic chemical.
Furthermore, diffusional characteristics are rarely considered in
encapsulation of microbial biological control agents. Co-encapsu-
lated low-molecular weight components such as drying protec-
tants and nutrients (e.g., trehalose, glycerol, glucose, calcium
gluconate) will with most methods diffuse out of the capsules dur-
ing the crosslinking. Resulting capsules often contain only about
half of the nutrients when applied. After reswelling diffusional
characteristics often imply that the rest of the substrate is washed
out of the capsules with the first irrigation or rainfall as is well-
known from diffusion coefficients of immobilized biocatalysts
(Buchholz et al., 2005).
Redissolvability is needed for applications where solid formula-
tions are to be released either immediately (e.g., in tank mixes for
spray applications) or for a slow release where the matrix is soft-
ened. This can be obtained by a change of pH, temperature, ion ex-
change, enzymatic reactions or reswelling. For example, ionotropic
hydrogels containing entomopathogenic nematodes will redissolve
in sodium chloride solutions (Patel and Vorlop, 1994).
Biological degradability in soil is important because many bio-
polymers are degraded too fast, especially in soil. The degradability
of biopolymers depends on various microstructural parameters
and the nature of the chemical bonding between the monomers
(Endres and Siebert-Raths, 2009). There are only a few systematic
studies about biodegradation processes of polymers and their gel
systems for applications in agriculture (Puoci et al., 2008; Vroman
and Tighzert, 2009).
For example, Ratajska and Boryniec (1999) tested films of a
two-component mixture containing synthetic and natural poly-
mers. Their results showed that the biodegradability in soil and
water depends on the dimensions of the natural component parti-
cles and their distribution in the film. Generally, biopolymers with
lower biological degradability should result in longer persistence
of capsules and therefore in longer persistence of the biocontrol
agent.
7. Outlook
In the past microbial biological control was content with
polymers and the respective gels serving as a simple encapsulation
matrix. Modern encapsulation science takes into account that the
materials can add function to capsules more than just being an in-
ert matrix based on their individual characteristics depicted in Ta-
bles 1 and 2. There is a great number of data on interactions of
polymers with their physicochemical environment (pH, T, salts,
sugars, solvents, CO2, light, electric current, magnetic fields. . .)
especially in the realm of the so-called smart polymers. But much
less knowledge is available on how the capsule matrix interacts
with the physicochemistry of soil or phyllosphere and even less
on how they interact with the agro-ecosystem. For instance, in
the case of chitosan, it is now known that the polymer exhibits fun-
gicidal activity. There is no reason why all the other polymers
available should not show direct or indirect effects on the agrobi-
ology. That is why in-depth interdisciplinary studies on formula-
tion materials in the system soil–plant-pathogen/insect pest-
biological control agents are needed.
While encapsulation science has moved to highly sophisticated
capsule systems, the standard capsule in biological control remains
the calcium alginate bead. In the near future, formulations will aim
to co-formulate several active ingredients in one formulation such
as two biological control agents, a biological control agent and a
chemical pesticide or a biological control agent and efficacy-
enhancing agents resulting e.g., in novel synergistic formulations
such as attract & kill capsules. Due to advances in polymer
387
sciences, novel multiresponsive co-polymer gels, which respond
to combinations of triggers, are to be expected. This will all have
direct impact on capsule morphology hopefully resulting in novel
intelligent multi-compartment capsules.
With regard to the methods, more effort should be put into
adapting already existing methods to production and application
of microbial biological control agents. This may include clever
combination of already existing methods and gelation principles
to solve some of the main problems of microbial biological control:
shelf life and targeted delivery.
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