BIOMAT., ART. CELLS & IMMOB. BIOTECH., 21(3), 299-306 (1993)

A NEW BIOENCAPSULATION TECHNOLOGY FOR MICROBIAL INOCULANTS

Bernard DIGAT INRA

Station de Pathologie v6gCtale et de PhytobactCriologie rue Georges Morel

49070 BEAUCOUZE

INTRODUCTION

I t is generally accepted that for microbial inoculants, the type of chosen formulation and delivery is a key-point for the efficiency of the inoculum. Liquid formula t ions are most easily obtained but they are not always satisfactory because storage and preservation are difficult (and subjected to possible contaminations). Solid formulations, dehydrated or lyophilized, such as powders, give occasionally better results and the life time of inoculum can be extended. However, one of the main problems is that microorganisms, before becoming active, have to be rehydrated, which causes generally a high loss in viability due to osmotic shock. These liquid or solid formulations set moreover the problem of the whole release of inoculums at once at the same time with the advantages but also the associated risks. Gelled formulations resolve in part the drawbacks previously described and, in this case, the used techniques consist in entrapping directly the microorganisms i n polymer gels. But the release of entrapped microorganisms is not a quick-acting mechanism and needs a complete degradation of alginate beads.

299

Copyright 0 1993 by Marcel Defier, Inc.

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300 DIGAT

The purpose of this study is to describe a new process of bioencapsulation which avoids these disadvantages allowing a true macroencapsulation and a faster release of microorganisms. This process is particularly well-adapted to obtain microbial macrocapsules.

MATERIAL and METHODS

In the process, the macocapsule included a core and an envelope, these two parts being individualized. Several types of microorganisms were encapsulated according to the process.

1. Core of the capsule and microbial inoculum Living microbial inoculum was exclusively concentrated in the core of the

capsule. Very different types of microorganisms such as bacteria, fungi, nematodes and viruses were encapsulated. Bacteria and fungi (Table 1) were grown in their liquid specific medium up to the stationnary phase and the concentration was adjusted to 109 CFU/ml. Entomophagous nematodes (Neoaplecrana capocapsa) were produced i n Galleria mellonella L. larvae, harvested, washed for 10 minutes in a Merseptyl bath (1 %O sodium ethylmercurithiosalicylate) and rinsed in sterile RINGER’S solution. The suspension concentration was about 5000 nematodes per ml and was stored at 10°C until encapsulated. Viruses (Tobacco Mosaic) were extracted from Nicotiana tabacum “White burley” infected leaves. Leaves were ground at + 4OC in a 0.002 M tris (hydroxymethyl) amino-methane-HC1 buffer, pH 7.2 to obtain an infective sap containing Tobacco Mosaic Virus (TMV) particles. The viscosity of microbial suspensions was CONTRAVES TV type and then adjusted up to 70 to 150 centipoises (cps) depending on the microorganism by adding a viscosing agent. One of the most frequently used viscosing agent was a micronised powder of Algae from horsetail help (Laminaria digitata L. Laminariaceae), gamma rays sterilized, at the rate of 9 % (w/v). The pH was adjusted to the neutrality.

2. Envelope of the capsule and coating The envelope material was made of a sterile mixture of 1.5 % sodium

Alginate and 10 % kaolin (w/v) in distilled water with a viscosity of about 350 cps. The reticulation agent of the envelope was a sterile saturated solution of Calcium

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BIOENCAPSULATION TECHNOLOGY FOR MICROBIAL INOCULANTS 301

TABLE 1. TYPES OF ENCAPSULATED MICROORGANISMS

MICROORGANISM

Bacteria

Fungi - myconizae

- yeast - non plant pathogenic

Nematodes

G E N U S , SPECIES, S T R A I N I NAME OF DONOR*

Pseudomonas fluorescenr L26- i Bradyrhizobium japonicum G 49

Laccaria lacuua Paxill us involurus

Saccharomyces cerevisiae Fusarium oxysporum FO 41

Neoapleciana carpocapse (= Sieinernema feltiae)

B. DIGAT J.C. C L A W - M A R E L

J. GAGNON D. STRULLU

%I. LESAFFRE c. A L A B o m

C. LAUMOND

* We thank the donors who supplied strains for this work.

gluconate. The capsule was finally coated with a thin pellicle of either a clay such as Bentonite that makes easy dehydration, either a hydrophobic agent such as Methyl- cellulose and by-products that avoids the sticking and packing of beads.

3. Equipment for encapsulation and making of capsules The head encapsulation equipment was composed of three elements A, B

and C disk-shaped of 10.5 cm diameter superposed and assembled in a leak-tight manner (Fig. 1) . The pasty system which was intended to form the core, anived under pressure via the central channel of an injector, forming a viscous drop at the end of the needle. Simultaneously, the material intended to form the envelope flowed into the annular space of the injector in form of drops which fell into a 0.1 M Calcium gluconate solution (Fig. 2). Several times of permeation in Calcium gluconate were tested. The capacity of equipment was determined by the number of impulses per minute of the pump. The external diameter and the volume of the core of capsules were depending on the diameters of the end nozzles of central and coaxial channels and might vary from 3 to 8 millimeters. For example, to obtain capsules with a 6 millimeters external diameter and a 110 pl core, the nozzle diameter of the central channel was 1.5 millimeter and the coaxial channel 3.0 millimeter.

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1 2

Fig 1. - HEAD OF ENCAPSULATION EQUIPMENT

1. Core channel 2. Envelope channel A, R, C, elements

1 2 3

Fig. 2 - STEPS OF ENCAPSULATION 1. Formation of the core by a viscous drop at the end of the

2. Formation of the envelope by flowing into annular space

3. Granules exit from injector

central channel

and surrounding the core granule

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BIOENCAPSULATION TECHNOLOGY FOR MICROBIAL INOCULANTS 303

T A B L E 2. CHARACTERISTICS* OF C A P S U L E S

Diameter (mm) 6

Weight (mg) 168

Volume (PI) 250

- envelope 140 - core 110

Residual hygrometry (%) 93

* These data represent the average calculated from lo00 granules

4. Dehydration a n d assessment of t h e survival of encapsulated

The survival of microorganisms i n the capsules V.S. residual hygrometry and dehydration time in a ventilated atmosphere at 40°C was studied.

The assessment of the survival of encapsulated microorganisms was studied by releasing them from capsules. For releasing, samples of one hundred capsules were immersed i n 100 nil of a sterile Phosphate buffer (pH 6.4, 0.02 M) and vigorously shaked at 4°C. After one hour, the capsules were wholly desintegrated and supernatant was investigated to defect and numerate the microorganisms according to the classical methods.

microorganisms

5. Induced degradation of the capsule envelope Several products able to induce the degradation of envelope were tested.

Among these, Ammonium phosphate, Ammonium carbonate and Sodium carbonate were chosen because their compatibility with microorganisms. Capsules were immersed in 1 M sterile salt solution and the degradation was visually observed during 24 hrs.

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DIGAT

RESULTS

1. Structure of microbial capsules and equipment capacity Characteristics of capsules are summarized in Table 2. In our conditions of

experiment, the mean volume of the core was 110 pl for a 6 millimeters external diameter of the capsule with five minutes permeation in Calcium gluconate. However at least 12 hrs permeation in Calcium gluconate were necessary to complete calcification and, after this time, the volume of capsule was slightly reduced. It was checked that capsules made from a bacterial inoculum of about 109 CFU/ml contained a mean of about lo8 bacteria. A slow rate of pump (1 impulse/second) and an equipment head with six injectors allowed more than three kilograms of capsules per hr to be made, i.e. about 20 000 capsules. I t was possible to increase the capacity by increasing the number of injectors and the number of impulses.

2. Effect of dehydra t ion on t h e su rv iva l of encapsu la t ed microorganisms

At 40°C. survival of encapsulated microorganisms V.S. dehydration time was correlated with residual hygrometry. It was observed that the survival was gradually decreasing with the residual hygrometry decreasing. For bacteria at this temperature, when the dehydration time exceeded fifty minutes, the bacterial survival and residual hygrometry obviously droped. A 15 % residual hygrometry was considered as a limit and induced a loss of about one power. For example, after 50 minutes of dehydration at 40°C of capsules containing 107 Pseudornonas fluorescens cells, more than 106 bacteria stayed alive at a 15 % residual hygrometry (Fig. 3). This significant dehydration resulted in a two fold decrease in capsule diameter. Finally the dehydrated capsules were kept in sealed flasks at 4°C for six months without any loss of bacteria.

3. Induced degradation of the capsule envelope In vitro degradation induced by ammonium and sodium carbonates was

extremely rapid. One hour after immersion of capsules in 1 M carbonate solution, the envelope was entirely degraded. In 1 M Ammonium phosphate solution, 24 hrs of immersion were needed to induce the complete degradation of the envelope.

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BIOENCAPSULATION TECHNOLOGY FOR MICROBIAL INOCULANTS 305

7.2

- 7,O a C

I 0

6,8 c 0

0 0 -I 6.6

6.2

6,O

100 - Log clu/granule - Residual Hygrometry YO $ r. L -

-80 2 - 0 % I

m -

- -60 2 v) al

m a - - - 40

-

8 I ' I I 1 I 20 I ' I I

0 1 0 2 0 30 40 50 60

Dehydratation time (mn) at 40°C

Fig. 3 : SURVIVAL OF BACTERIA IN GRANULES

DISCUSSION and FUTURE PROSPECTS

This new encapsulation process resulted in a high concentration of microbial inoculum in a small volume, for example up to 109 Pseudomonas cells per capsule. Because microorganisms were suspended in their specific nutrient medium and then embedded with algae extract, the osmotic shock and the nutritional stress were avoided specially during the releasing phase. Algae extract seemed to be a well- adapted medium for microbial survival. In the specific case of Pseudomonas fluorescens, one of the key factors in survival was the preservation of a 15 %

residual hygrometry in capsules but strain resistance to temperature stress could also be a survival factor because of the demmental effect of high temperatures which are frequently required for dehydration (1).

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306 DIGAT

New applications of the process are in progress ( 2 , 3) to be adapted to microalgae and plant embryo encapsulation. Indeed, in this last case, envelope and core of the capsule could mimic respectively teguments and albumen of the seed and it would be possible to develop artificial seeds. The capsules, being entirely biodegradable in soil, are environmentally compatible and would contribute to forming a microbial inoculuni for agronomic applications such as plant-growth promotion, biocontrol of plant pathogens or degrading pesticides and fertilizers refuses i n soil.

Finally. the system offers the possibility of encapsulating simultaneously two types of microorganisms. For example, bacteria can be included in the envelope and fungi in the core or vice-versa.

REFERENCES

1. DIGAT B m d MATTAR J., 1990. Effects of temperature on growth and siderophore production of Pseiidomonas fluorescens-purida strains. Symbiosis 9 : 27-213.

2. DIGAT B., 1987. Structure for including active substances. lnfernafiorialpafenf

3. DIGAT B., 1990. Granules h multicouches contenant des substances actives enrobCes ; leur procCdt5 de fabrication ; dispositif pour la mise en oeuvre de ce procCdC et utilisation des granules. European patenf PCT n"90/00604.

n o W088108876.

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