bioencapsulation of microbial cells for targeted agricultural delivery

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Introduction

Soil quality is totally affected by the application ofchemical nitrogen fertilizers and exceeding applicationbeyond the crop requirement creates many environmen-tal problems in all ecosystems (Mulvaney et al., 2009).One of the solutions for eliminating the negative effect ofchemical ammoniacal nitrogen fertilizer is crop rotation with rhizobial symbiotic leguminous plants. Inoculationof leguminous plants using pure cultures of Rhizobium isa common practice (Ben Rebah et al., 2007). According toPerret et al., (2000), the practice of rhizobial inoculationbecame promising after the discovery and application ofnitrogen fixing bacteria by Hellriegel and Beijerinck in

the 1880s. According to Guthrie (1896), Beijerinck firstisolated and grew rhizobia and used them in legumes. Inearlier days, farmers transferred the fertile soil to othersterile fields to make them suitable for agricultural useand the transfer of the soil (after leguminous plant growth)to other agricultural fields was considered as the primaryphase of plant inoculation technology. In the early twen-tieth century in Canada, seeds were treated with soil

containing nodulating bacteria and sticking agents beforesowing ( Walley et al., (2004). According to Bashan (1998),inoculation with non-symbiotic nitrogen fixing bacteriain large scale production was practiced in Russia duringthe 1930s and 1940s. According to Bashan (1998), rhizo-bial inoculants were produced around the world for yearsinitially by small companies. In countries such as Braziland Argentina there is no application of chemical nitro-gen fertilizer for legumes cultivation, such as soybeanand hence they are inoculated with rhizosphere bacteria(Dobereiner et al., 1995). As Bashan (1998) reported, the1970s witnessed major advancements in plant inocula-tion technologies such as the invention of nitrogen fixing

bacterium (e.g. Azospirillum) and biocontrol agents (eg.Pseudomonas spp.). Later, various other bacterial genera were also evaluated for plant growth promotion (Bashan, 1998). Nowadays, inoculation of seed with the desiredmicroorganisms and soil application of these microbesare considered as two inoculation technologies. Directseed inoculation has a positive effect only with sufficientmoisture content. Moisture is critical since desiccation

REVIEW ARTICLE

Bio-encapsulation of microbial cells for targeted agriculturaldelivery

Rojan P. John1, R.D. yagi1, S.K. Brar1, R.Y. Surampalli2, and Danielle Prévost3

1INRS-ETE, Québec, Canada, 2 USEPA, Kansas City, Kansas, USA, and 3 Agriculture et Agroalimentaire Canada,Sainte-Foy, QC, Canada

AbstractBiofertilizers, namely Rhizobium and biocontrol agents such as Pseudomonas and Trichoderma have been wellestablished in the field of agricultural practices for many decades. Nevertheless, research is still going on in the field

of inoculant production to find methods to improve advanced formulation and application in fields. Conventionallyused solid and liquid formulations encompass several problems with respect to the low viability of microorganismsduring storage and field application. There is also lack of knowledge regarding the best carrier in conventionalformulations. Immobilization of microorganisms however improves their shelf-life and field efficacy. In this context,microencapsulation is an advanced technology which has the possibility to overcome the drawbacks of otherformulations, results in extended shelf-life, and controlled microbial release from formulations enhancing theirapplication efficacy. This review discusses different microencapsulation technologies including the productionstrategies and application thereof in agricultural practices.

Keywords: Biofertilizer, encapsulation technology, microencapsulation, shelf-life, controlled release

Address for Correspondence : R.D. yagi, INRS-EE, Québec, Canada. el.: 418-654-2617; Fax: +1-418-654-2600; E-mail: [email protected]

(Received 13 April 2010; revised 22 July 2010; accepted 02 August 2010)

Critical Reviews in Biotechnology , 2011; 31(3): 211–226

© 2011 Informa Healthcare USA, Inc.ISSN 0738-8551 print/ISSN 1549-7801 onlineDOI: 10.3109/07388551.2010.513327

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212 Rojan P. John et al.

Critical Reviews in Biotechnology

occurs at lower moisture and the germination of seedsrequires high moisture, thus formulation development is very important.

Bioinoculant formulation

According to Xavier et al., (2004), formulation is a vitalcharacteristic for bioinoculant development of an effec-

tive microbial strain and it can decide the functionalsuccess or failure of a biological agent. Formulation usu-ally consists of the microorganism and a suitable carriertogether with additives. Additives along with carrier, aidin the stabilization of formulation and protection of themicrobes from environmental influence during storageand transport. It helps microorganisms to perform betterat the application site ( Xavier et al., 2004). According to Xavier et al. (2004), a formulation should be stable duringproduction, storage, and shipping. It should be easy tohandle and apply in order to give better performance ofinoculation. Besides, all the formulations should be cost-effective ( Xavier et al., 2004, Jones and Burges, 1998).

Tere are numerous effective solid and liquid formu-lations, such as peat based powders or suspensions, andemulsions for agricultural applications. However, the lackof availability of good carriers, environmental impactsand factors affecting the biological agents regarding for-mulation lead to the search for improved formulations.Te potency of the formulation is critical for the success-ful commercialization of the bio-inoculants products.Tus, formulation is a challenging and often successlimiting step in the successful commercialization of newbio-inoculants. Tis necessitates the development ofstable biofertilizer or biocontrol agent formulations fromalternative raw materials which can compete with the

existing inoculants as well as the chemical alternatives.Research is still ongoing to find an efficient formulationfor agricultural applications and this review focuses onthe bio-encapsulation aspects of microorganisms for thedevelopment of efficacious biofertilizers and biocontrolagent formulations.

Conventional formulation versus advancedformulation

Common carriers in conventional formulation According to Stephens and Rask (2000), conventionalbiofertilizer or biopesticides are used in plant applica-tions as powder, liquid or granulated forms. Powderand liquid inoculants are generally applied to the seeds(preinoculated seeds), while granular inoculants areapplied to the soil (to seed furrows or soil mixing) (Ben Rebah et al., 2007). Inoculants are usually commercial-ized as solid and liquid inoculants (Stephens and Rask, 2000). Powder or granular inert materials may includeplant growth media or matrices, such as rockwool andpeat-based mixes, attapulgite clays, kaolinic clay, mont-morillonites, saponites, mica, perlites, vermiculite, talc,carbonates, sulfates, oxides (silicon oxides), diatomites,

phytoproducts, (ground grains, pulses flour, grain bran, wood pulp, and lignin), synthetic silicates (precipitatedhydrated calcium silicates and silicon dioxides, organ-ics), polysaccharides (gums, starches, seaweed extracts,alginates, plant extracts, microbial gums), and derivativesof polysaccharides, proteins, such as gelatin, casein, andsynthetic polymers, such as polyvinyl alcohols, polyvinylpyrrolidone, polyacrylates (Date and Roughley, 1977;

Dairiki and Hashimoto, 2005; Jung et al., 1982).

Need for alternative carriersEarlier, seed coated peat-based inoculants dominated thecommercial inoculant market due to the positive impactof peat on the storage and application processes. Duringsoil inoculation, peat based formulations can alter the soilstructure and can provide a new protective habitat for therhizobial cells ( van Elsas and Heijnen, 1990). Althoughpeat was recognized as a very good carrier of rhizobia,there was interest in developing alternate formulationsas its availability was not widespread and due to its nega-tive environmental impact on peat available ecosystems

(Hynes et al., 1995). According to Albareda et al., (2008),the liquid inoculant production process is simpler asthere is no alteration of carrier and the application of theformulation is easier. Liquid formulations include sus-pensions, concentrates, and oil based products, such asemulsions. However, bacterial survival is inferior in theliquid type of inoculants and on liquid inoculated seedas bacteria lack carrier protection ( Albareda et al., 2008;Singleton et al., 2002; ittabutr et al., 2007). Alternativeformulations were developed to obtain the advantage ofa good shelf-life by additive supplementation and easytransportation by decreasing the volume, which mini-mized the handling steps (John et al., 2010). Terefore,

the adoption of these formulations reduced the overallassociated cost compared to other conventional for-mulations. Recently, some advanced technologies havebeen developed for the effective storage; transportation,and enhanced efficiency of formulations by encapsulat-ing rhizobial cells using polymers.

Encapsulation technology

Encapsulation is the technique of making a protectiveshell or capsule around the active ingredient or cells (e.g.microbial, macrobial cell or tissue).

Advantages of cell encapsulation According to McLoughlin (1994), the matrix of microbeads protects the inner cells from both mechanicalstress and the adverse conditions of the outer environ-ment by providing a defined, constant, and protectivemicroenvironment. Te cells can survive and metabolicactivity can be maintained for extended periods of time, with controlled release of cells after their adaptation tothe surrounding environmental conditions (Cassidy et al., 1996). Microencapsulation can noticeably improve the viability of microorganisms due to its protective effects

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Bio-encapsulation of microbial cells 213

© 2011 Informa Healthcare USA, Inc.

against detrimental environmental factors, such as pH variation and the poisoning agents generated duringthe process (Mortazavian et al., 2007). It can protect thebacterial cells from bacteriophages, hydrogen peroxide,short chain fatty acids, carbonyl-aromatic compounds,and drying (Mortazavian et al., 2007). Te immobi-lized cells showed tolerance to alcohols (Holcberg and Margalith, 1981). Tis tolerance was mainly due to the

enhanced modification of the cell membrane (Groboillot et al., 1994). Alginate contains many fatty acid impuritiesand these impurities probably result in a modified fattyacid pattern for the immobilized cells compared withfree cells (Diefenbach et al., 1992). A similar effect of tol-erance against phenol in E. coli was reported (Keweloh et al., 1990) and it was attributed to the uptake of thesaturated fatty acids present in commercial alginates andincorporation into the cellular membrane. Later, Keweloh et al. (1990) suggested that it was not only fatty acids butalso other ingredients of alginate that had a physiologicaleffect on the cell membrane (Diefenbach et al., 1992).

Te release of microorganisms from micro beads is

also a simpler but controlled process. A micro-envelopemay be opened by many different means, includingfracture by heat, solvent action, diffusion, and pres-sure (Brannon-Peppas, 1997). Tus, the encapsulatedmicroorganisms possess much more efficiency thanthe conventional powder and liquid formulations. Bothconventional and advanced formulations have their ownmerits and demerits as illustrated in able 1.

Macro and microencapsulationTe encapsulation of cells is of two types: macroencapsula-tion and microencapsulation. Macroencapsulation is theentrapment of cells in polymeric structures of a larger size

extending from few millimeters to centimeters. Usuallyanimal cells or tissues are entrapped by this method tomake artificial cells or tissues. Macroencapsulation canbe defined as encapsulation by surface coating materi-als, such as polymeric organics (e.g., resins and plastics)or inert inorganic materials to substantially reduce thesurface exposure to potential leaching media (http:// www.setonresourcecenter.com/cfr/40CFR/P268_030.HM). According to Bashan (1998), the use of macroalginate beads in agricultural application has two majordisadvantages and these are; additional treatment duringsowing and the need for the bacteria to move through thesoil towards the plants. Tis problem with macro-beadscan be solved by the use of micro-beads as they can havedirect contact with the seed. According to Lin and Chen (2002), micro beads have a size of 10–100 µm and macrobeads are greater than 100 µm. Te encapsulated mate-rial can be rounded beads, cubes or even sheaths.

Microtek laboratories has defined microencapsulationas the process of surrounding or enveloping one sub-stance within another substance on a very small scale, yielding capsules ranging from less than one micron toseveral hundred microns in size. Te core materials willbe released either slowly through the capsule walls by

diffusion or when external conditions prompt the capsule walls to burst, melt or dissolve (http://www.microteklabs.com/technical_overview.pdf ). Te bigger sized particles,such as microencapsulated cells are usually releasedby the latter method. Te macro-encapsulation of seedinoculants has less uniformity when mixed with seed.From an application view point, the microencapsulationgives uniformity in spreading, is effective and results in

an easy release of microorganism to the targeted sitedue to the small size and higher surface area. Te loss ofmicrobial cells during the preparation of microbeads isminimal compared to large beads (Bashan 1998). It canbe easily applied to soil using sprayers or can also bedirectly coated on seeds.

Microbeads and their structure According to Park and Chang (2000), microbeads areencapsulated microbial cells and consist of hydrogels(capsule) coated around the microbial cell or cells. Teshape, size, and texture of the beads vary with the typeof coating material and microorganism employed. Te

shape may be spherical, elliptical or irregular and containone or more cells (Figure 1). Te filamentous core mate-rial like fungal fragments or actinobacteria can vary inshape as they have different shapes and size. Te coatingof the beads in the case of multilayered beads are durablethan other single coated gel beads. Te ultrastructure ofmicrocapsules revealed a micro-reservoir structure in which the wall extended well into the core and the activeagent (e.g. microbial cells), and was accommodated bythe micro-reservoirs (Figure 2). Most of the materialsused in encapsulation are polymers, either naturallyoccurring (e.g. polysaccharides, proteinaceous material)or synthetic (e.g. Polyurethane foam) (Groboillot et al.,

1994, Saucedo et al., 1989). Te polymers may be homopolymers (e.g. gelatin) or hetero or co-polymers (poly-lactic acid-polyglycolic acid). Te polymers are selectedon the basis of the chemical composition of the mono-meric units. Te quantity and characters of the functionalgroups contained in the monomer units define the pri-mary structure of polymers. Te chemical composition ofthese monomer units gives rise to various interactions,such as hydrogen bonding, electrostatic interactions, andhydrophobic interactions, which are important for intra-and intermolecular interactions (de Vos et al., 2009) suchas gelling or cross-linking.

Besides the chemical composition of polymer, themolecular weight also has significant role in the encap-sulation. According to Lu and oy (2009) one of themajor drawbacks of polyethylene glycol (PEG) was itshigher molecular weight. Te loading level of PEG wasinversely proportional to its molecular weight as it hasfunctional groups only at the terminals of the polymerchain. Te optimum molecular weight of PEG in dis-tearoyl-N -(monomethoxy poly(ethylene glycol)succinyl)phosphatidylethanolamine (DSPE–PEG) was 2000 anda larger molecular weight resulted in lower encapsula-tion (Moribe et al., 1999). Jalil and Nixon (1990) tested

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poly(DL-lactic acid) having three different molecular weights (20500, 13300 and 5200) to prepare microcap-sules comprising phenobarbitone, as a reference coreusing the water/oil emulsion evaporation method. Jalil and Nixon (1990), observed a trend towards loweringthe mean microcapsule size, both by volume and popu-lation was observed with respect to a lower molecular weight polymer. With variations in polymer molecular weight, no effects were observed on the morphology ofthe microcapsulated surface, efficiency of encapsulation,and density (Jalil and Nixon, 1990).

Techniques of microencapsulationMethods of encapsulation vary with the nature of appli-cations from diverse fields for miscellaneous objectives.Some techniques require incorporation of the support-ing material along with the central core cells for stability.Te formulation can be freeze dried or made into a liquidemulsion and a suspension to assimilate the components.Further, these formulations can be micronized, micro-emulsified or coated for further modification and finallypolymerized, gellified, spray dried or coacervated to pro-duce stabilized micro particles. For further protection,

Capsule

Microbial cell

5

4

3

1

2

Figure 1. Different forms of microbeads: (1) spherical, (2) oval,(3) irregular surface, (4) multicellular, and (5) multilayered bead.

Lipophilic layer

Active ingredient

Hydrophilic layer

Figure 2. Diagrammatic representation of a microcapsuleshowing core and shell (capsule).

Table 1. Microbial formulation: conventional versus advanced types.

Conventional formulation Advanced formulation

Solid formulation Liquid formulation Encapsulated cells

In solid formulation, the shelf life ofmicrobes is poor or average due todesiccation. Tis is not only in thestorage period but also in the fieldapplication.

Shelf life is poor or average due to theosmotic imbalance and lack of oxygen

More compared to both conventional methodsdue to the reduced influence of environmentand easy accessibility of nutrient and oxygenthrough simple diffusion.

Te cost of raw material and production

cost is lower but unavailability of the bestmaterial is a limiting factor.

Te cost of material and process are

higher compared to solid formulation.

Te cost of the polymeric compound is higher

than the other easily available solid and liquidformulation components but the higher costtypes can be replaced by low cost gum orgelling agents of biological origin.

Tere is the need of more cells in theformulation development and fieldapplication. Washing off percentage andmortality is high.

As in the case of solid formulations the viable biomass necessity is high. Erosionand mortality of cells are higher due tothe lag period in adaptation.

Te viability of microbes is higher in the caseof encapsulation. Controlled release to theexternal environment reduces the wastage ofmicrobial cells. It helps the microbes to adaptto the environment by giving protection.

Contamination by other microbes ishigher. Antimicrobial agent is thusmandatory in solid formulations.

A contamination problem exists even inthe case of emulsions but less comparedto solid. Antimicrobial agent is essentialin some liquid formulation and willnegatively affect the microbial survival.

Te contamination of antagonistic organism is very less due to the protective shell or sheathoutside.

ransportation is costly due to the bulk

size, but some dry formulations such asspray drying with some fillers or freezedrying agents increase the cost ofproduction.

Concentrated microbial emulsion and

suspension will decrease the size ofstorage vessels and transport is easierbut each step increases the productioncost.

Minimum storage space is required as it is

immobilized in micro or macro beads as thelow volume application and thus reduces theinitial fermentation cost.

Tere is less bridging of seeds as theformulation is dry.

In seed applications, there is the need oflarge volumes to produce the minimumlevel of cells and there is clumping manyseeds.

Te use of microencapsulated formulation islow so there is no clumping of two or moreseeds.

No or less need for sophisticatedequipment.

No or less need of sophisticatedequipment.

For microcapsule preparation there is theneed of equipment relating to spray andsolidification.

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there can be utilization of more polymer coating basedon the application and types of beads and organisms. Alginate beads can be hardened by the post treatment with chemicals, such as gluteraldehyde (Park and Chang, 2000). According to Park and Chang (2000) polyvinylalcohol could be treated with boric acid solution to makethe bead strong and long-lasting, but a prolonged crosslinking process reduces the survival of the cells.

Different methods dealing with microencapsulationare summarized in Figure 3. Te important methodsused for encapsulating microorganism are discussed inthe following text. Research has also been carried out toincrease tolerance using other additives, such as, oil andpoly-L-lysine or certain methods, such as cross linking orcoatings (Ding and Shah, 2009; Ghosh, 2006; Hyndman et al., 1993). Te first step of encapsulation is the disper-sal and integration of microorganism into a liquid matrix.Tis is followed by a selective process that can be used forthe production of micro-beads or a microcapsule whichdepends on the organism or encapsulating materials orapplication. Among the methods mentioned in Figure 3,

spray drying and extrusion are the two major commer-cial processes usually used in terms of product volume.Te important methods are described in the subsequentsections.

ExtrusionExtrusion is a very old and simple method of makingcapsules with hydrocolloids (King, 1995). According toKrasaekoopt et al. (2003), the extrusion process could bedone by mixing the microorganism with hydrocolloidsfollowed by extrusion into a hardening solution. Te sizeand shape of the beads could vary based on the distanceof free-fall and the size of needle pore. Tus, the extruder

die plays an important role in the extrusion process. Teflow, deformation and temperature relationships shouldalso be considered while designing an extruder die(Sokhey et al., 1997).

A pumping system can be utilized to make uniformand small sized beads. In this case, the pressure flow (Q)mainly depends on the viscosity of the liquid. Accordingto Liang (2001), viscosity is a crucial parameter for the flowcharacteristics of polymer fluids. Liang (2001) reported

that Hirai and Eyring (1959) proposed a relationshipbetween viscosity and pressure as follows, based on theEyring hole theory of fluid,

η = η β −P P

P P 0 0 0

exp ( )

(1)

where, ηP and η

P 0 are the viscosity at pressure (P ) and

atmosphere pressure (P 0), respectively. β is the pres-

sure coefficient which is a function of the hole volume

and absolute temperature. So, extrusion pressure mustbe regulated on the basis of the viscosity of the fluid fora better encapsulation and for stable bead formation.Sokhey et al. (1997) tested the effect of die measurementon the extrusion performance on food products usingmaize. According to Sokhey et al. (1997) “die diameteraffected the radial expansion of the extrudate but axialand overall expansions were not affected by die diam-eter. Length of the die had no influence on radial expan-sion of bead but had a major effect on axial and overallexpansions. During extrusion, operating pressure isinversely proportional to increasing die diameter butdirectly proportional with die length. For a constant die

length, increase in operating pressure increased radialexpansion. A die with small diameter and shorter lengthshould be used for greater radial expansion and mini-mizing energy consumption”. Te real diameter of thebead is again corrected by shrinkage or swelling factors.Usually alginate beads tend to shrink during gelation while a nylon microcapsule swells on washing (Dulieu et al., 1999).

Emulsion technique Water-soluble materials are widely used for applicationsin agriculture, food, and pharmaceuticals (Baogou et al., 2006) and generally a water-in-oil emulsion has beenused to make micro-beads. Krasaekoopt et al. (2003) described emulsion technique as follows; a small volumeof the cell-water soluble polymer (e.g. alginate, gelatin)suspension is added to a large volume of a vegetable oil.Te mixture is homogenized to form a water-in-oil emul-sion and the polymer in emulsion cross-linked to formtiny gel particles within the oil phase. Te final size of themicro-particles varies according to type of emulsifica-tion, depends on the internal phase particle size of theemulsion. Te cross-linking method depends on the typeof polymer support (e.g. gelatin can be cross-linked usinggluteraldehyde or alginate can be gelled by calcium ions).

Te size of the beads is controlled by the speed of agita-tion. McNamee et al. (1998) used gum arabic for makingemulsions with soya oil and the mixture was spray driedfor microencapsulation. Te selection of gum arabic wasdue to its high solubility, low viscosity compared to otherhydrocolloids, and ability to act as an oil-in-water emul-sifier. Later, McNamee et al. (2001) replaced gum Arabic with other carbohydrates.

Spray drying According to Picot and Lacroix (2003), microencapsula-tion by spray drying is a well-established process that can

Coacervation

Microencapsulation Technologies

Droplet freezing

Polymerization

Interfacial

polycondensation

Supercritical fluid

Droplet gelation

Thermal gelation

Gelation

Extrusion

Solvent

evaporation

Spray-drying

Fluidized bed

Figure 3. Different processes in microencapsulation technology.

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produce a large quantity of material. Tis technology isnot considered as a good cell immobilization techniquedue to a high mortality resulting from simultaneousdehydration and high temperature inactivation of micro-organisms. Picot and Lacroix (2003) micronized thepowder particles produced by spray drying and emul-sification methods, using a spiral jet mill as a grindingsystem. According to Picot and Lacroix (2003), the effect

of micronization on cell viability was mainly influencedby the final particle size. Micronization was determinedto be an efficient means of reducing the particle size offreeze-dried culture powders with a satisfactory deathrate before producing microcapsules with low diametersfor the protection of heat sensitive bacteria (Picot and Lacroix 2003).

In accordance with Ghosh (2006), the fermented brothcan be quickly dried after mixing in a polymer solutionand sprayed into a hot chamber. Te in-feed temperatureis most often kept at or close to ambient temperature forthe viability of microorganisms, such as non-sporulatingRhizobium. Some other sporulating bacteria and spores

of fungus, such as Trichoderma sp. can withstand an evenhigher drying temperature. High pressure spray nozzleand centrifugal wheels can make very minute particlesand it can reduce the exposure time of heat-labile micro-organisms. Te particle size distribution of microencap-sulated bacteria may be limited to a lower range thanthe given particle size in conventional formulations. Tehypothetical curve of particle size distribution is shownin Figure 4. Te lower size of microbeads helps in theirbetter performance. Te major limitation of the spraydrying process is the mortality of microorganisms asmentioned above, along with the damage to the shellby micro-cracks that can lead to decomposition of the

capsule. Te process costs may also be high with the useof sophisticated equipment, power consumption, andexpensive carriers.

Amiet-Charpentier et al. (1998b) reported microen-capsulation of Pseudomonas fluorescens-putida usingspray-drying. No bacteria were alive at time zero, when

the inlet air temperature was at 80°C for the micro-spheres harvested in the cone and flask. While at 60°C,there was bacterial survival and it was estimated thatnearly ~107 CFU/g of micro-particles were obtained inthe cone, after 2 months of storage. At time zero, it wasnoticed that the residual moisture of the microspheresdecreased for the particles collected both in the flaskand cone, when the inlet air temperature was increased

from 60 to 80°C. Tus, it is clear that desiccation dueto higher temperature destroys the microbial cells andprotective measures have to be taken for better survival. A similar result was observed in the case of biocontrolfungal ( Beauveria brongniartii ) conidia of encapsula-tion, when the outlet temperature was raised to nearly53°C ± 2°C so that the viability decreased (Horaczek and Viernstein, 2004).

Amiet-Charpentier et al. (1998b) reported that feedrate also influence the survival of microorganisms dur-ing spray drying. A lower spray feed rate decreased themicrobial survival due to a faster desiccation rate and vice versa. At a spray feed rate of 3–4 mL/min, there was

no bacterial survival at time zero. By doubling the rate to7–8 mL/min, at time zero bacterial survival of ~104 CFU/gmicro-particles was obtained in the flask. Bacterialsurvival of ~107 CFU/g was obtained in those collectedin the cone. Tis bacterial survival was continued to bethe same after 2 months of storage in the encapsulatedform. Tis increase in survival was due to the increase inresidual moisture when the spray feed rate was doubled( Amiet-Charpentier et al., 1998b).

CoacervationTis is mainly used as a technique for controlled drugrelease, and the process relies upon the decrease in

solubility of coating polymer by the addition of anothercompound to the polymer solution, changing pH or tem-perature (Park and Chang 2000). Microencapsulation bycoacervation can be achieved by the phase separation ofthe coating polymer solution. Polymer rich coacervate wraps around the liquid core and the solvent supernatant

Figure 4. A hypothetical curve for particle size distribution (PSD) for a normal liquid versus the microencapsulated formulation. (PSDdata derived in our laboratory as tested on a liquid formulation).

8

Frequency distribution

Cumulative frequency distribution

Hypothetical

microencapsulation profile

7

6

5

4

F r e q u e n c y

d i s

t r i b u t i o n

3

2

1

0 0

20

40

60

C um ul a t i v ef r e q u en c y d i s t r i b u t i on

80

100

120

0.1 1 10

Particle size (µm)

100 1000

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separates, finally leading to the solidification of the micro-particle (Nihant et al., 1995). Tis method is not suitablefor producing lower sized microspheres and there is for-mation of harmful residues on the microspheres. Use ofsupercritical gases as phase separating agents can mini-mize the formation of these harmful residues (Freitas et al., 2005).

Solvent extraction/evaporationSolvent extraction/evaporation is a simple method ofmicroencapsulation to achieve a controlled particlesize. Tis method does not require high temperaturesor inducing agents for phase separation (Freitas et al., 2005). Vigilant choice of encapsulation materials andconditions and a low residual solvent content in the finalpreparation will give particle sizes in the nano to microm-eter range (Freitas et al., 2005). According to Freitas et al. (2005), solvent extraction/evaporation basically consistsof four major steps: (i) dispersal of the microorganism orother active ingredients in a solution of matrix formingmaterial; (ii) emulsification of this phase in a successive

continuous immiscible phase; (iii) extraction or evapora-tion of the solvent and transforming microglobules intosolid microspheres; (iv) harvesting and drying of themicrospheres. Te temperature for evaporation is not sohigh in this process and is tolerable to the microorgan-isms. But the mortality of the microorganisms is mainlycaused by the solvent used in this process which may belethal to the microbe. Sometimes, higher concentrationof the solvent can desiccate the microbial cells and a lon-ger duration of the process may affect the viability of theorganism. Tus, solvent extraction/evaporation may notbe a viable technique for formulation of the biologicalcontrol agent.

Thermal gelationTis is another promising technique in cell encapsulationand can be achieved by cooling a warm aqueous polymersolution. Beads are obtained by dropping the polymersolution in cold water or oil. Te cell-polymer solutiondropped into media containing a cross-linking agent was suddenly cooled from 40–45°C to 25°C to gelate themicro-beads after strong vortexing in the reactor ( Audet et al., 1989). Autoclaved Κ-carrageenan at 40–45°C wasmixed with cells and dropped into a cold KCl solution. oobtain better results, the polymer can be emulsified with warm oil and cooled by adding cold oil or by using heatexchangers (Neufeld et al., 1991).

Pregel dissolving technique According to Park and Chang (2000), the double steppregel dissolving technique was developed by Lim andSun in 1980. Te sodium alginate containing cells aredropped into a CaCl

2 solution and the calcium alginate

beads are made by a conventional method. Ten thecarboxyl group of the calcium alginate bead are allowedto react with an acid-reactive amine or imine group in acompound such as, poly-L-lysine or polyethyleneimine.

A polyelectrolyte complex membrane is formed on thesurface of the calcium alginate bead and the bead isretreated with sodium alginate solution to crosslink theresidual poly-L-lysine on the surface of the bead. Tesolid core capsule of calcium alginate is dissolved insodium citrate solution and forms a liquid core with themicrobial cell (Park and Chang, 2000). Tis preparationis impractical in agricultural application as there was no

microbial release for an extended time due to the highstability of the beads. It can be used for the industrialproduction of organic acids or other compounds usinganaerobic or micro-aerophilic bacteria.

Factors affecting microencapsulatedmicrobial cells

Te selection of suitable capsule materials that are ableto remain stable in the surrounding environment is very important. Each and every material has differentfunctional groups, monomer differences and an abilityto interact with other components during microencap-

sulation. A capsule should have sufficient mechanicalresistance to withstand the various forces during theentire duration of production and application, espe-cially in bioreactors. Mechanical stability does not havemuch importance during agricultural applications asthe capsule has to degrade or dissolve for controlledrelease into the environment. However, resistance ofmicrocapsules is very important during storage andtransportation.

Alginate is considered as the most common biomate-rial in the encapsulation of bacterial and/or other type ofcells (Murua et al., 2008). Commercial sources of alginateare brown algal species especially Laminaria,Macrocystis,

and others (Cheze-Lange et al., 2002). Te natural alg-inate polymer is formed by linking D-mannuronic acidand L-guluronic acid monomers at the C-1 and C-4 posi-tions (Smidsrod and Skjak-Braek, 1990). Alginates createthree dimensional structures when they react with mul-tivalent ions and it is the basis of its use as a gel matrix. According to Murua et al. (2008), the gelling of the aque-ous alginate solutions is due to the binding of divalentcations between the G blocks of adjacent alginate chainsand creating inter-chain bridges. A natural material likealginate is not harmful to the survival of microorganisms.Common capsular material used in bioencapsulation ispresented in able 2 and the advantages, disadvantagesand possible modifications of important materials areincluded in able 3.

Te coating material of the capsules and type ofinterstitial fluids are two other parameters that affectthe efficiency of microencapsulated cells by modify-ing the physicochemical characteristics of beads. As inpregel dissolving, the hardening material of the surfaceof the alginate bead helps to increase the hardness andincrease the leakage of cells. But, the coating materialcan influence the material transfer in reactor applica-tions and affect the survival of cells. Te number of

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entrapped cells in each bead is directly proportional tothe concentration of microbial cells in the encapsula-tion solution and increased concentration of the cellsin beads directly influence quantitative efficiency ofencapsulation. Te quality of microbial cells, such as tol-erance to pH and temperature, sporulation ability, floc-culation, exopolysaccharide production, antimicrobialactivity, and additive tolerance also affect the survival of

encapsulated organisms (Sultana et al., 2000). Termalgelation is the best method for thermopilic organisms, while pregel dissolving is suitable for anaerobic organ-isms. As mentioned earlier, the factors during microen-capsulation process, such as freeze drying, spray drying,micronization, and storage conditions are very impor-tant to avoid the mortality of contained cells and injuriesto the beads (Sultana et al., 2000). Te bead size and cellload are critical parameters for encapsulated bacteriaduring storage and application.

Akin to the physical forces in the microencapsula-tion process, the additives used to maintain the viabil-ity of microorganisms or to maintain the properties of

the microcapsule also have great influence on the per-formance of microorganisms in microbeads. Additives

are the substances that aid in the stabilization andprotection of the microbial cells in a formulation dur-ing storage, transport, and at the target zone ( Xavier et al. 2004). Different additives which aid in the stabil-ity and survival rate can be applied during the forma-tion and storage of micro-beads. A study comprisingrhizobacteria revealed that bacterial survival washigher with the seed coating agent, Sepiret 1039G, and

rapid release of the microencapsulated bacteria wereshown using wettability measurements of agglomer-ated micro-particles ( Amiet-Charpentier, 1999). Tephysical-chemical characteristics of the coating mate-rial showed that water was the critical parameter forthe release of rhizobacteria.

As mentioned above, the formulation must be stablefrom production to application site, it should enhancethe activity of the organism in the field, be cost-effective,and be practical ( Young et al., 2006). Te additive isone of the major cost factors in various formulations. Along with carrier, additives play a crucial role in aprolonged survival during different stages of formula-

tion. Many commonly available materials were usedas additives according to the types of application and

Table 2. Different carrier materials used in encapsulation of microbial cells.

Carrier Microorganism Use Reference

Urea-formaldehyde Mixed culture Crude oil, Hexadecane,Phenanthrene degradation

Mohn, (1997)

Agar-alginate Yarrowia lipolytica Crude oil degradation Zinjarde and Pant, (2000)

Polyurethane Yarrowia lipolytica Crude oil degradation Oh et al. (2000)

Gellan gum Mixed culture Gasoline degradation Moslemy et al. (2002)

Alginate Prototheca sp. n-Alkanes degradation Suzuki et al. (1998)

Azospirillum brasilense Cd Biofertilizer Bashan, (1986)

(ACC 29710) Weir et al. (1995)

Pseudomonas sp. Phenanthrene degradation Paje et al. (1998)

Rhodococcus spp. Benzene degradation Sheu and Marshall, (1991)

Lactobacillus delbrueckii ssp. Ice cream production

bulgaricus

Polyacrylamide Pseudomonas putida Benzene degradation Somerville et al. (1977)

Alginate, agar,polyacrylamide

Pseudomonas spp. Naphthalene degradation Manohar andKaregoudar, (1998)

k-carrageenan andlocust bean gum

Streptococcus thermophilus Yoghurt production Audet et al. (1988)

L. casei ssp. casei Biomass production Arnaud et al. (1992)

Chitosan, Cross-linked with Hexamethylenediisocyanate or glutaldehyde

L. lactis ssp. cremoris Biomass production Groboillotet al. (1993)

Gelatin, cross-linked withtoluene-2,4-diisocyanate

L. lactis ssp. cremoris Biomass production Hyndman et al. (1993)

Gelatin–polyphosphatepolymer

Pseudomonas sp. Biocontrol agent Amiet-Charpentier et al. (1998a)

Xanthan and locustbean gum

Bradyrhizobium japonicum

Biofertilizer Jung (1980)

Eudragit®, methacryliccopolymer

Pseudomonas fluorescens-putida

Biofertilizer Amiet-Charpentier et al. (1999)

Eudragit®, methyl cellulose,modified starch

Pseudomonas fluorescens-putida

Biofertilizer Amiet-Charpentier et al. (1998b)

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microorganisms. Vassilev et al. (2001a) used skimmedmilk and clay as additives to enhance the performanceof gel entrapped soil microbial inoculants during soilapplication. A controversial effect was observed with thesurvival of P-solubilizing bacteria on application of char-coal-soil mixed with alginate ( Viveganandan and Jauhri, 2000). Young et al. (2006) showed humic acid along withalginate was the best additive in the encapsulation of

rhizobacteria to enhance field performance. It is a chal-lenging task to incorporate a suitable additive along with the active ingredient without causing any nega-tive impact on cell. Generally, the mortality of the cellsmay be higher if they are directly applied to the soil, andadditives help to adapt with the environment soon afterthe application. For example, materials, such as humicacid can influence the soil dwelling organism for accli-matization to the soil environment. Materials, such asgelatin, skimmed milk or whey protein may provide thenutritional requirements of entrapped microorganisms.

Encapsulation of microorganisms for

agricultural applications

Soil fertility and plant growth is greatly influenced bymicrobial assistance in the rhizosphere as microbialpopulations in the soil are involved in fundamentalactivities that ensure the stability and productivity ofagricultural and natural ecosystems (Barea et al., 2005).Te industrial sector in agriculture has mainly focussedon the isolation and commercial cultivation of thesemicroorganisms for high yield and disease preventionin an environmental friendly manner. Researchers havedeveloped many formulations of these microorganismsto improve crop production and recent research focus

on advanced methods to improve formulation efficiencyduring storage and application.

Bacterial applicationIt is well established that introduction of plant growth pro-moting bacteria to soil improves plant growth. Bacteria,such as Pseudomonas fluorescens-putida, act as a plantgrowth stimulator and biocontrol agent (Bashan 1998).Some bacteria can fix nitrogen by symbiotic association with leguminous plant root. Bacteria that are able to nod-ulate leguminous plants can be classified into five genera,such as Azorhizobium, Bradyrhizobium, Mesorhizobium,Rhizobium, and Sinorhizobium. Some plants, such asPhaseolus vulgaris can be a host for many bacteria to nod-ulate the plant. Te important bacteria which nodulateP. vulgaris areR. leguminosarum bv.phaseoli (Jordan, 1984),R. etli (Segovia et al., 1993), R. tropici ( Martinez-Romero et al., 1991), S. meliloti , R. leguminosarum bv. trifolii (Michiels et al., 1998), and R. gallicum bv. phaseoli andR. giardinii bv.phaseoli ( Amarger et al., 1997). Meanwhile,others are host-specific.

Rekha et al. (2007) reported that Pseudomonas putida CC-FR2-4 and Bacillus subtilis CC-pg 104 produced asignificant effect on the shoot height of Lectuca sativa

L. seedlings. Bacteria encapsulated in beads had moreeffect than free cells after 21 days of growth and thusthe authors claimed that the entrapped bacterial cellscould be a novel and feasible technique for applicationin the agricultural industry. Te soil application of bac-terial beads at sowing time is one of the frequently usedmethods for encapsulated microorganisms either as abiocontrol or a biofertilizer. Many encapsulation studies

have been reported with bacterial beads application toagriculture and beads were made up of polyacrylamide(Dommergues et al., 1979), or polysaccharides, such asalginate (Diem et al., 1988; van Elsas et al., 1992), carra-geenan (revors et al., 1992; Bashan, 1986) and xanthan(Jung et al., 1982; Mugnier and Jung, 1985), among others.Tese bacterial beads were produced by co-extrusion ofan alginate solution to obtain spherical granules of 6 mmdiameter (Digat, 1988) or by rhizobacterial macroencap-sulation (Jung, 1980). Fertilizer formulation, comprisingrhizobacteria has also been encapsulated in a calciumalginate matrix (Fages et al., 1988). Tese methods pro-duced only larger encapsulated beads or material and

did not suit the seed industry, which was searching fordirect seed inoculation. Bashan (1986), described directbacterization of wheat seeds with beneficial rhizospherebacteria. Te study found that the macroencapsula-tion of seed with alginate-containing bacterial cells wasimpractical due to a storage incompatibility betweenthe bacteria and seeds, as the seeds need a dry environ-ment to avoid germination during storage, but bacterianeed more moisture for their survival. As the size of thebeads is very big and almost the same size as the seeds,there is no uniformity in the distribution of large scaleseed mixing. Direct bacterization of various seeds usingrhizobacteria (about 108 bacteria per seed) in calcium

alginate in a double inclusion technique was perfprmedby Digat (1991) and more than 107 bacteria per seedremained alive after 1 h at 40ºC. Callan et al. (1991) stud-ied the field performance of maize seeds coated with aPseudomonas fluorescens AB254 suspension in a methyl-cellulose against a disease caused by Pythium ultimum.Te treatment provided an equal effect of fungicide inall conditions that were tested. Te major drawback ofpreinoculated seeds is undesired seed germination dur-ing storage, due to the high water content of the appliedformulation. In addition, drying processes after seedcoating with formulations cause water loss leading to themortality of the microorganism due to desiccation.

Amiet-Charpentier et al. (1998a), has recounted thatdehydration during storage prevented germination ofseed with high moisture beads by fabricating cross-linked complex coacervated gelatin-polyphosphatebeads. A suspension of bacteria in paraffin oil was usedto make water-in-oil (w/o) emulsion, where the internalaqueous phase contained the bacteria and there was nodirect contact with seeds. Tus, a double, water-in-oil-in-water (w/o/w) emulsion was formed in the aqueoussolution of gelatin and cross-linked with gluteraldehyde.Tis approach should make it possible to keep bacteria

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in an aqueous internal phase within the polymer micro-particles. According to Amiet-Charpentier et al. (1998a),the drawback of this system was that it was not goodfor the survival of aerobic bacteria as the air-transfer was reduced. Tis method can be successfully used forthe development of dead bacterial formulations andanaerobic bacterial formulations ( Amiet-Charpentier et al., 1998a). Some reports found that polymers can

provide much longer viability of up to one year. Amiet-Charpentier et al. (1998b) tested three polymers, namelymethacrylic copolymer, ethylcellulose, and modifiedstarch for the microencapsulation and spray drying ofa plant growth promoting bacteria. Eudragit®, meth-acrylic copolymer was found to be better for the survivalof Pseudomonas fluorescens-putida. A silica additive wasalso examined for enhancing the moisture retention andsurvival of bacteria. In this test there was no effect ofsilica on the moisture content but it enhanced the survival of bacteria at time zero, however during storage, theeffect was negligible. Amiet-Charpentier et al. (1998b) stated that it might be the result of the larger particle size

of silica gel than the bead as it is situated outside the gel,and absorbed water from the bead to the outside of theadditive particle. So, the additive particle has to be com-patible with the microencapsulated particle.

Amiet-Charpentier et al. (1998b) found a relationshipbetween bacterial survival and the residual moisture ofmicrospheres. Te best survival of the stored bacteria was observed at ∼25% of the residual moisture. In anotherstudy, Amiet-Charpentier et al. (1999) used Eudragit® as a formulation which was the first report on bacterialinoculant and proposed in a micro-particulate form.Te survival of the bacterial cell in micro-particles wasstudied under different levels of relative humidity, such

as 0, 33, 55 and 100%. Bacterial survival was satisfactoryfor the formulation at 100 % relative humidity. Te pres-ence of silica in the formulation improved the bacterialsurvival when the relative humidity becomes between 55and 33% ( Amiet-Charpentier et al. 1999). Compatibilitystudies were performed between film-forming agentsand the encapsulated bacteria. Compatibility washigher with one of the two film-forming agents (in thepowder form, Sepiret® and an aqueous dispersionform, Cerprotecteur®), for the seed coatings tested andimmobilized bacteria were released when in contact withexcess of water ( Amiet-Charpentier et al. 1999).

Similar to biofertilizer application, bacterial agentscan also be encapsulated for their use as biocontrolagents. A simple strategy is the application of biocontrolagents such as bacteria, Bacillus thuringiensis. Attemptsto create autoencapsulated formulations of B. thuringi-ensis against European corn borer, were made by mixingstarch powder and sugar (McGuire et al., 1990; Shasha and Dunkle, 1989). Bok et al., (1994), suggested the useof carbohydrate rich biopolymeric gels for constructionof matrices, and they observed a 50% mortality of thediamondback moth even after two weeks. Te entrappedbiopesticide in biopolymeric gels (potato starch, rice,

rye, barley, and soybean powders) could retain itsentomocidal activity through better protection againstdessication, sunlight, heat, and the damaging effects ofUV light (Bok et al. 1993). Cote et al. (2001), reported anextended period of mortality of Choristoneura rosaceana with a bioencapsulated formulation of B. thuringiensis var. kurstaki in comparison to conventional Dipel™ (aregistered Bacillus thuringiensis var. kurstaki (Btk)-based

formulation). Among the tested meteorological factorssuch as, precipitations, temperature, and solar radiations,precipitation was more predominant as if reduced thepersistence of the insecticidal activity (Cote et al. 2001).

Fungal applicationMost fungal parts can be used as mycopesticides, but allof them are susceptible to environmental parametersand there is worldwide acceptance for these formula-tions. Fungi belonging to the genera Conidiobolus,Entomophaga, Nomuraea, Paecilomyces, Tolypocladium,Verticillium, Entomophthora, Erynia, Neozygites, Pandora,Hirsutella, Metarhizium, Aschersonia, Beauveria etc. are

well known as biocontrol agents against many pests (Shah and Pell 2003). Trichoderma sp. was used as a biocontrolagent against many fungal pathogens. Many strains ofTrichoderma have been investigated and considered aspotential biocontrol microorganisms. Trichoderma viride ACC 52440, for example, is able to produce biocontrolagents against soil borne plant pathogens. Tis funguscan be used for the biocontrol of Fusarium oxysporum,F. solani, Phytophthora capsici, Sclerotium cepivorum,and Verticillium dahliae (Cho and Lee, 1999). Generally,formulations of the biocontrol strains of Trichoderma are used in the form of alginate pellets containing theirspores (Knudsen and Bin, 1990; Lewis and Papawizas,

1985), and spores and hyphal segments in gluten (Choand Lee, 1999).

Akin to rhizobacteria, mycorrhizal fungi play differentroles in the soil nutrient improvement and plant growth,such as phosphate nutrition, plant water potential underdrought stress, bioprotection against various patho-gens and improvement of soil structure, among others( Vassilev et al., 2005). According to Vassilev et al. (2005),there are at least 1350 combinations of natural, semi-syn-thetic, and synthetic polymers for the entrapment of bio-materials but the majority of techniques involved in situ entrapment by using natural polysaccharide gels whichinclude alginates, agar, and carrageenan. According to Vassilev et al. (2005), the first successful encapsulation ofmycorrhizal fungi was performed by Ganry et al. (1982), with endomycorrhizal fungi. Horaczek and Viernstein (2004), examined a spray drying mechanism for encap-sulating Beauveria brogniartii for the formulation againstMelolontha melolontha. Te study on fungal encapsula-tions in skim milk and polyvinylpyrrolidone mixturematrix, pointed out that spray drying of the fungal aerialconidia was highly viable after spray drying even thoughthere was a decline in viability during an increase in theoutlet temperature. According to Horaczek and Viernstein

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(2004), the survival of fungal material was inverselyrelated to the storage temperatures and residual mois-ture levels of the spray dried powders. Te inlet-outlettemperature must be selected based on the type of aerialmaterial and the storage of bioencapsulated materialmust be carried out at low temperature with minimummoisture. Each and every part has a different toleranceand usually the conidia or other thick walled spores are

used for prolonged storage and long duration during for-mulation production.

Mixed culture applicationo attain different results together, various biologicalmaterials belonging to several genera with synergisticaction can be applied simultaneously. Cowpea treated with Glomus mosseae (entrapped in Ca-alginate beads)could improve nitrogen fixation with applications along with Bradyrhizobium (Ganry et al., 1985). Vassilev et al. (2001a) co-entrappedG. deserticola containing root piecesand cells of the P-solubilizing yeast Yarrowia lipolytica in alginate gel. Tey pointed out that the introduction

of an entrapped inoculant formulation into soil-plantsystems enriched with insoluble inorganic phosphatesshowed that plant dry weight, soluble P-acquisition, andmycorrhizal index were higher than the treatments withfree and Ca-alginate entrapped G. deserticola includedin the presence of free cells of the yeast inoculant. Asimilar study with G. deserticola and G. fasciculatum revealed that the introduction of both inoculant formula-tions into soil for the growth of the tomato plant as a testshowed average levels of root colonization between 26%(G. fasciculatum) and 32% (G. deserticola) which werefurther enhanced to 39% and 49%, respectively, whenthe mycorrhizal fungi were co-entrapped in the alg-

inate beads with Y. lipolytica (Vassilev et al., 2001b). Another experiment by Vassilev et al. (2001c) showed afour times higher nodule number was observed whenGlomus deserticola (an arbuscular mycorrhizal fungus) was introduced into a soil-plant system with Rhizobiumtrifoli and the results were compared with the controlplant nodulated only with R. trifoli . Addition of Y. lipoly-tica with a soil-plant system enhanced root mycorrhizalinfection by about 14%. Co-encapsulated R. trifoli and Y.lipolytica yielded a ten fold increase in root nodulation inthe tested plants ( Vassilev et al., 2001c).

Effect of natural agents on formulation performanceTe effect of sunlight is crucial in foliar applicationbesides eroding factors, such as rain. Sunlight, mainlydirect heat and UV rays play an important role in themortality of biopesticides. Even though encapsulatedbacteria or fungal material have more tolerance thanfree cell formulation, the application of heat tolerantsubstances in encapsulating material can drasticallyreduce the effect. According to Brar et al. (2006), vari-ous UV radiation screens, such as Congo red, folic acid,molasses, lignin, alginate, cellulose, shellac, and p-amin-obenzoic acid were tested for UV protection and showed

mixed results. Protein-based polymers, such as glutenand casein are advantageous for use as coating materialsas they are biodegradable, harmless to the environment,cheaper, and readily available. Protein coatings preventthe UV exposure of prepared biological materials that aresensitive to UV light ( Yu and Lee, 1997). Selection of heattolerant bacteria or fungal material such as spores can bemore effectively utilized in tropical areas than the easily

desiccating biomaterial.Rainfall is another important natural agent that wouldaffect the persistence of biopesticides on foliage leadingto wash-off and thus a reduction of the efficiency of thebiopesticide (Behle et al., 1997). Wash-off has a nega-tive effect not only on foliage application but also on soilapplication. Encapsulation can reduce microbial wash-off by rain fall or runoff water. When the bacteria wereencapsulated, the wash down rate was reduced comparedto free inoculated bacteria, as most of them remained inthe root zone (controlled release) ( van Elsas et al., 1991;1992). P. fluorescens embedded in alginate beads had thepotential to produce a biocontrol effect against bacte-

rial wilt of tomato by its colonization on the root ( Aino et al., 1997). revors et al. (1992) proposed that bacterialinoculation using alginate resulted in good rhizospherecolonization and survival on the plant, a low wash-downrate, and resistance to drying. Corn starch formulationshowed a higher efficiency after rainfall due to the stick-ing property of the starch (Brar et al., 2006). Gelatinizedor modified starch sticker addition can also enhance theefficiency of encapsulated biopesticides by applicationon leaf surfaces.

Tere are several other factors, such as bead size,shape, and cell load that have to be taken into account while applying on to foliage or other arial parts of the

plant. Papavizas et al. (1987) studied the effect of wheatbran on the proliferation of biological material, such ashyphae, conidia, and ascospores of Talaromyces fIavus. Conidia and ascospores showed rapid proliferationbut hyphae did not propagate. In contrast, Lewis and Papavizas (1985) reported some food base addititives thatcan increase the hyphal proliferation of Trichoderma andGliocladium in alginate beads. Tese facts indicate thatthe selection of organism, type of encapsulation material(bacterial cell, spores, and mycelia), encapsulating agent,additives, and others as key parameters for the encapsu-lated formulation with respect to their application.

Future prospects and recommendations

Due to the benefits over conventional formulations,encapsulated formulations can be recommended forthe agricultural applications of microorganisms, bothfor soil and aerial applications. Among bioencapsulatedforms, microencapsulated formulations are preferableto efficient applications such as foliar spraying or seedcoating. Seeds of plants like alfalfa are very small andonly microencapsulated forms can be coated on suchseeds for a uniform distribution. Te seed coating of

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microencapsulated formulation is cost effective and uni-form as a minimum volume of the formulation is usedfor seed application and the bioinoculant can have aneasy access during the emergence of radicle or plumule.Microencapsulated formulation can be applied easily onseeds or soil-like liquid formulations after the requireddilution. In microencapsulated beads cells are highlyconcentrated and the volume of these formulations is

very low; therefore these formulations can be stored in alimited space, transported easily, and diluted many timesduring applications.

Te risk of a biocontrol agent in the foliar applicationis higher compared to soil applications as there is theinfluence of environmental parameters, such as sunlightand rain fall. However, using an effective means of bio-encapsulation along with proper screens and stickers would enhance the effect of biocontrol activity. Besides,the additives and the biomaterial selection are also veryimportant depending upon the geographical area andmode of application. Microencapsulation of bacteriaand fungal spore is easier than the fungal mycelia due to

their microstructure. Te fungal thick-walled spores pos-sess higher tolerance than the mycelial fragments. Tus,spores can be utilized for the effective production ofmicroencapsulated material than the mycelia, as it helpsproduce a uniform distribution. Generally, actively grow-ing fungal mycelia are more active in biocontrol thanapplied spore formulations as they can grow faster in thesoil. Te beads stored at low temperature (the survivalrate is very high under refrigerated condition) can bebrought to the optimum growth temperature to enhancegermination, and hence increase the performance on theapplication site. For soil applications, actively growingmycelia can be entrapped and these macrobeads can be

mixed with soil.Emulsion techniques can be adopted for microencap-

sulation of agro-friendly microbes and other techniquessuch as spray drying or solvent extraction for the superiorsurvival of thermo-or chemo-liable bioinoculants. In ourprevious work, it was shown that emulsion formulationis better for the concentrated bioinoculant productionand it could enhance the survival of microbes duringprolonged storage (John et al., 2010). Hence, the encap-sulated beads can be kept as such in oil after production,emulsified with suitable emulsifier, or separated from theoil phase for further formulation developments. A biore-actor can be used for the production of microbeads withthe emulsion technique and thus agitation and feeding ofingredients can be controlled.

Our group has proved the use of different agro-indus-trial waste and wastewater for efficient and economicproduction of biocontrol and biofertilizing microorgan-isms. After harvesting and concentrating, the microbialbiomass along with the other ingredients (unutilizedpolymeric substances or hydrolysed substrates) in themedium can be used for the micro-encapsulation.Usually the sludge contains di- or trivalent metal ionssufficiently in its natural form and this innate character

can be explored for the self encapsulation by mixingpolymers, such as alginate. Te concentrations of thesemetal ions are sufficient for supporting their growth andthus it will not affect survival. Other by-products such asbrewery sludge, powdered brewery spent grain or hydro-lyzed spent brewing yeast can be supplemented to theharvested microorganisms. Te selection of additives inpolymer must be performed carefully based on the type

of microorganism and its role on the growth or survivalof microorganism. Te cost effectiveness can be achievedby replacing skimmed milk (a general additive) with whey concentrate or whey powder. Te use of these lowcost materials can also act as carbon source for microor-ganisms during storage and applications. Biodegradablesynthetic polymers are a good option with respect to costeffectiveness, survival of microbes, and environmentalimpact. Natural gums, such as locust bean gum, xanthangum and gum arabic can be applied as stickers, anti-desiccant or gelling agent, and thus the formulation canbe completely environmentally friendly.

Te particle size of the additive must be smaller than

the prepared beads; otherwise the microorganism haslower accessibility to the additive. Te bigger size ofthe additives has a negative influence on the survival ofmicroorganisms due to the moisture extraction outsideof the bead. Te selection of microencapsulation tech-nologies, such as emulsion technique and spray dry-ing and also the process parameters, such as durationof formulation development, temperature, and curingtime must also be taken into consideration while usingthe desired bacterial or fungal material. As in the case ofspray drying, the inlet air temperature, drying durationin the column, and the storage temperature in the coneare critical.

Te disadvantages of microencapsulation technologymust not be ignored. Many of the microencapsulationtechnologies require special equipment. In the desiredprocesses, such as spray drying, several sequential stepsare needed for proper encapsulation. Microencapsulationduration is longer than the preparation of other formu-lations. Te material cost used for the encapsulationis generally higher than other formulations. Terefore,abundant and economically feasible materials shouldbe adopted. Biofertilizer and biopesticides have a majorimpact in agricultural applications due to their eco-friendly nature. Environmental effects must also be takeninto consideration while selecting the material and pro-cesses for microencapsulation.

Te market for biocontrol agents and biofertilizer varies with different countries as there are varied rulesand regulations for application of microbes. Tere hasto be a universal regulation and acceptance for bioin-oculant applications and both environmentalists andthe government have to take initiatives for testing andcommercialization of these biological and eco-friendlyformulations. Consumer anxiety about live microbes isanother limiting factor for commercialization. Propereducation and awareness for solving these issues and

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support from financial agencies (public and private) hasto be implemented.

Conclusions

Different solid and liquid formulations have been devel-oped for the application of microbial biofertilizer orbiocontrol agents in agriculture, albeit with advantages

and disadvantages. Te emerging field of microencap-sulation is a promising step to obtain the controlled andeffective release of these microorganisms for targetedagricultural applications. Te importance of the modifi-cation and testing of these formulations is necessary forthe cost effective and environmental-friendly productionand application of this system. Most of the current stud-ies report positive effects of utilizing these advanced for-mulations. Furthermore, exploitation of different types ofmatrices of biological origin, effectiveness of encapsula-tion of each and every microorganism for agriculturaluse, survival during storage and application is necessaryto overcome the drawbacks of conventional solid and

liquid formulations.Bacterial microencapsulated formulations for soil and

seed application are superior to other formulations as itenhances the survival of bacteria, and controlled release with prolonged effect. Even though the cost of productionis slightly higher, microorganisms in microencapsulatedformulation are recommended due to their higher per-formance during storage and applications. It is expectedthat in future, efficient microencapsulated formulations will conquer the market for better agricultural productionand to protect the environment from non-degradablechemical pollutants.

Declaration of interest

Te authors would like to thank the Natural Sciences andEngineering Research Council of Canada (Grants A4984,Canada Research Chair), MAPAQ (807150). One of theauthors, RPJ, would like to thank the Fonds Québécois dela Recherche sur la Nature et les echnologies (FQRN),for the postdoctoral fellowship under the programme“Programme de bourses d’excellence pour étudiantsétrangers”.

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