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Alailableonlineatwww.sciencedirect
com
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Contlol
EI,SEVIER Biological Control 3l (2004) 65-71
www.elsevicr.com/locatdvbcon

Comparisonof threecommonlyuseddrying technologieswith respect

to activity and longevity of aerial conidia of Beauveriabrongniartü
and M etarhiziumanisopliae
Andrea Horaczekand Helmut Viernstein*
Institute of Phamuceutical Technologyord Biophannaceutics,Centerof Pharmacy, Unioersityof Vienna,Althotst. 14, A-1090 Vienna, Austria

Received9 October 2003;accepted27 April 20O4

Available online l5 Junc 2üX
ffi
Availableonlineat www.sciencedirect.com
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dor^..ro Biological
Contlol
EISEVIER BiologicalControl 3l (2004)65-71
www.elsevier.com/locate/ybcon

Comparisonof threecommonlyuseddrying technologieswith respect

to activity and longevityof aerialconidia of Beauueriabrongniartii
and Metarhiziumanisopliae
Andrea Horaczekand Helmut Viernstein*
Institute of PharmaceuticalTechnologyand Biopharmaceutics,Centerof Pharmacy, Uniuersityof Vienna,Ahhanstr. 14, A-1090 Vienna,Austria

Received9 October 2003;accepted27 April20o4

Availableonline 15June2004

Abstract

We comparedthreecommonly useddrying technologiesfor preservationand formulation of 14-to 20-day-oldaerial conidia of

Beauaeriabrongniartüand,Metarhiziwn anisopliae.Conidia were dispersedin an aqueousbinary mixture composedof skim milk
(SM) and polyvinylpynolidone (PVP K90) and subsequentlysubjectedto heat stresstests,lyophilization,and spray-and fluid-bed
drying.The initial heat stresstestsrevealedthat M. anisopliaeis slightly more heatresistantin the suspendingmediathan 8. brongni-
arlii toleratingexposureto 50'C for 2 min without lossin viability. In comparingthe drying technologieswith respectto germina-
tion rates,lyophilization was the most favorablefor 8. brongniartii and most destructivefor M. anisopliae.Yiabilitylevelsof 68 and
4% resultedafter freeze-drying.The influenceof differentinlet/outlet temperatureadjustmentsduring spray- and fluid-bed drying
wereexaminedaccordingto their influenceon conidial viability. Both drying methodsresultedin severedamageto the fungal mate-
rial alreadyat an inlet/outlettemperatureregimeof 60/40+2oC, reflectedby low and prolongedgerminationrates.
@ 2004ElsevierInc. All rights reserved.

Keywords:Entomopathogenic
fungi;Beauteriabrongtiartil,Metarhiziumanisopliae;
Freeze-drying;
Spraydrying;Ftuid-beddrying

l. Introduction developmentsin solid state culture systems(Bradley

Beauoeria brongniartü (Saccardo) Petch and Meta-
rhiziumanisopliae(Metchnikoff) Sorokin play an impor-
tant role in suppressingscarab beetles (Melolontha
melolonthaL. and PhylloperthahorticolaL.), and weevils
(Otiorhynchusspp.and Strophosomaspp.)(Burges,1998;
Ferron, l98l). Scarabbeetlesand weevilsare a major
threat to agriculture, forests, and horticulture (Butt
et al., 2001).The larvaefeedon the roots of a wide range
of economicallyimportant crops causingseveralbillion
Eurosdamageannually.
Severaldifrerentstagesof the fungal life cycle (coni-
dia, mycelial fragments or blastospores)are potential
candidatesfor mycoinsecticides.In nature, the aerial
conidium is the infectious and dissemination unit,
moreover,the increasein production efficiencydue to
' Correspondingauthor. Fax:.+43-l 4277 -9554.
E-mail address (H. Viernstein).
: helmut.viernstein@univie.ac.at
et al., 1992), make aerial conidia the propagules of
choice for the developmentof a biological insecticide.
For biological control, it is important to keepthe active
unit in an infectiousyet dormant stage,safe,and easyfor
application.The dormancyof conidiais exogenous, not
constitutive,so the key to prolonging their survival is to
stop germinationand to reducemetabolismas much as
possible.One possibilityfor stabilizationis dehydration.
Our objectiveswerethe preservationand formulation of
both entomopathogenicfungal speciesby three com-
monly used industrial drying methods. Lyophilization
and spray-and fluid-beddrying are amongthe most use-
ful techniquesfor preservingfoods, agricultural prod-
ucts, and pharmaceuticals(Hieda and lto, 1973;Nath
and Satpathy,1998;Teixeira et al., 1995).Biological
materials,however,can be irreversiblydamagedduring
these treatmentsdue to heat and bulk water removal
(Daemenand van der Stege,1982).To preventor reduce
these adverseeffects,our initial experimentsinvolved

1049-96441$ - seefront matter @ 2004ElsevierInc. All rights reserved.

doi:10.l0l 6/j.biocontrol.2004.04.0l6
 A. Horaczek,H. ViernsteinI BiologicalControl3l (2004) 65-71
heat resistancetests for the fungal isolates.Moreover,
protective substanceswere added to samples before
processing.The protectant fluid used in the present
investigationconsistsof skim milk (SM) and polyvinyl-
pyrrolidone (PVP). SM was chosenas it is a well known
membranestabilizerduring cryopreservation,an emulsi-
fication agent, and UV-protectant (Burges, 1998). In
addition, Teixeira et al. (1994)showedthe protective
effectof SM during heat treatment.The secondcompo-
nent of the carrier matrix, PVP, has been reported as a
protectivemedium for catalaseand other biological sys-
tems(Ashwood-Smithand Warby, 1912)and for human
erythrocytessubjectedto freezingand thawing (Morris
and Farrant, 1972).Furthermore,PVP is a very useful
formulation excipient,as it providesbinding power,film
formation, acts as an excellentbulking agent,and is vis-
cosity-increasing (Bühler,2001).As the sameprotectants
and temperatureregimeswere applied,the technologies
used can be directly comparedas to their influenceon
conidia viability and longevity,reductionof germination
speedand residualhumidity of the products.
2. Material andmethods
2.1. Fungalisolatesandmaintenance
Metarhizium anisopliae,isolated from Cydia pomo-
netla (L.) in Denmark and ,8. brongniartü,isolatedfrom
pasturesoil infestedwith M. melolonthq(L.) in Austria,
were both donated as monosporecultures by Dr. Her-
mann Strasserof the Institute of Microbiology of the
Leopold-Franzens University, Innsbruck, Austria.
Metarhizium anßopliaewas grown on Sabouraud'4Yo-
glucose (S4G medium; Merck; Darmstadt, Germany)
agar and B. brongniarriion Sabouraud-2o/vglucose (S2G
medium; Merck; Darmstadt, Germany) agar at 25oC in
the dark, respectivelY.
2.2. Preparationoffungal dßpersions for heat resßtance
pr -
studies, s ay dry ing, Iy op hiliz at ion, andfluid-b ed dry ing
Conidia suspensions werepreparedby flooding14-20
day cultures with sterile 0.1% (w/v) aqueousTween 80
(Merck; Darmstadt, Germany). For each experiment
and its repetition,a separateplate was used.The result-
ing conidial suspensionswere sonicatedfor 3 min and
vortexedfor I min to ensurehomogeneity.Conidial con-
centrations were determined using a Thoma hemocy-
tometer. Conidial concentrations were adjusted to
6.1x 107and2.5x 107for B. brongniartüand M. anisop-
/lae suspensiohs, respectively.An aqueousbinary system
composedof 5oÄ(w/v) SM (Merck; Darmstadt, Ger-
many) and 1.25o/o (w/v) PVP K90 (Fluka; Buchs,Swit-
zerland) was used as the dispersingand encapsulation
agent for conidia during heat resistance,spray-drying,
and lyophilization experiments.To increasethe binding
power of the aqueousbinary system,the proportion of
PVP was increasedto 5% (w/v) during fluid-bed drying
experiments. SM formulationswere sterilizedat 120'C
and I bar for l5 min, whereasthe PVP formulationswere
asepticallypreparedwith sterilizedwater as the polymer
was degraded by the high temperaturesrequired for
thermal sterilization.All aqueousformulationswerepre-
pared from demineralizedwater. Conidial suspensions
of both isolatesweremixed in the ratio of l:9 with the
aqueous carrier matrix system. The polymer/fungus
compatibility wasverifiedby germinationtests.
2.3. Heat resistancetests
Test tubes containing 900pl of the dispersingmedia
were exposedto the following test temperatures40, 45,
50, 55, and 60oC in a thermomixertype 5436(Eppen-
dorf; Hamburg,Germany).After temperatureequilibra-
tion occurred, measured by a thermometer, 100pl
conidial suspensionwas added and homogenouslydis-
tributedat l00rpmmin-1.After0.5,1,2,4,8, and l5min
at the temperatureused,the sampleswere removedand
cooleddown in an icy water bath for approximately30s
to reach ambient temperature.The germination of the
stressedconidia was investigated.For each speciestests
wereconductedat eachtemperaturein triplicate.
2.4. Spray-drying
The fungal dispersions were spray dried with a
laboratoryscalespraydryer B-191(Büchi;Flawil, Swit-
zerland)equippedwith a 0.7mm nozzleby adjustingair-
flow to 700Nlh-land aspiratorsettingto its maximum
valueof 35m3h-1. Sampleswereprocessed at inlet tem-
peraturesof60,80, and l00oC.Flow rateswereadjusted
accordingto the inlet temperaturesto resultin tolerable
outlet temperaturesfor the fungi. Furthermore, the
product temperaturewas monitored with a non-contact
temperaturemeasurementdevice,RayngerST3 (Raytek;
Berlin, Germany).The influenceof processparameters
on the germinationand on moisturecontent were inves-
tigated.Only microcapsulesprecipitatedinto the collect-
ing flask weresubjectedto germinationand storagetests.
Spray-dryingexperimentswere conducted in triplicate
for eachspecies.
2.5. Freeze-drying
Vials were filled with 500pl of conidial suspension
produced as describedabove and kept at -80oC for
24h. Following the freezingstep,sampleswereput into a
freeze-dryermodel CHRIST BETA 1-8K (Christ;Oste-
oC and
rode am Harz, Germany) operating at -35
0.090mbarvacuum for 20h. Secondarydrying took
placeat 0.090mbarvacuumwhile raisingthe tempera-
 A. Horaczek, H. ViernsteinI Biological Control 3l (2004) 65-71

ture gradually to ambient temperatures.The germina- Norwalk, CT, USA) to measurethe residualmoistureof
tion was checkedafter the freezingand drying process. the formulations. The residual moisture was obtained
Freeze-dryingexperimentswere conductedin triplicate after heatingfrom 20 to l00oC at 5 oCmin-I, holdingfor
for eachspecies. l0min at l00oC and subsequently heatingfrom 100to
130'Cat l0oCmin-r.
2.6. Fluid-beddrying
2.10.Data analysis
Fluid-bed drying was carried out with a laboratory
fluid-bed dryer model STREA-I (Aeromatic; Buben- Survival levelswere expressedas the quotient of ger-
dorf, Switzerland)equippedwith a 0.8mm nozzle.Inlet minated conidia before (N6) and after (N,) treatment.
temperatureswere adjusted at 60 or 80oC. Lactose Viability=(N1/N6)x 100. Means*SD were calculated
(Kwizda; Vienna,Austria) actedas carrier material with basedon data from triplicate experiments.
the addition of 5% (w/w) of a lubricant,a mixture of Mg-
stearateand talcum (l:9; both pharma grades).Ten mil-
liliters of the conidial suspensionswere sprayed onto 3. Results
l00g of carrier material.The developmentof the outlet
temperaturewasmonitored. Furthermore,product tem- 3.1. Heat resistancetest
peraturewas measuredwith a non-contacttemperature
measurementdevice,RayngerST3.The granulationpro- The testsrevealedthat M. anisopliaeis slightly more
cesswasperformedin triplicate for eachspecies. heat resistantin the suspensionmedia than -8. brongni-
artii (Fig.l).
2.7. Determinationof conidialgermination Difrerences in the temperature tolerance became
apparent at increasingexposureintervals and tempera-
Formulated dry conidia weredispersedin 0.1%aque- tures.Comparedto the germinationof freshly harvested
ousTween80 (Merck; Darmstadt,Germany).As control conidia, M. anisopliaedid not lose viability exposedto
acted untreated conidia washedoff 14- to 20-day cul- 40 and 45oC for l5min, whereasa declinein germina-
tures. Subsequently,50 pl of the resulting suspension tion rates occurred for B. brongniartii after 4min at 40
were spreadon agar plates (l% glucose,0.50Äpeptone, and 45oC. Thus, l5min exposureto 40 and 45oC
l.5oÄ agar, and 0.5oÄ yeast extract, amended with
3Omgl-r of streptomycinsulphate and 50mgl-l of A
chloramphenicol)and incubated for 24h at 25oC. The - 10o
:a
viability of 300 conidia was assessed
after staining with Eso
lactophenolcotton blue usinga Nikon l04light micro-
scope at a magnification of 400x. Only conidia with € r
germ tubes longer than their width were consideredto 5oo
have germinated.Survival levels were expressedas the E
E
ro
quotient of germinatedconidia before (tr[o)and after
(Nr) treatment.Viability:(N1/N) x 100. (,b o

Tlme(mln)
2.8. Determinationof storagestability
B
Spraydried powder and granulesproducedat an inlet F100
temperatureof 60oC and lyophilizates were stored at c 8 o
difrerent temperatures(6, 20, and 30oC). To the spray
dried powder formulation, silicagel capsuleswereadded
t*
e40
o
to keep the humidity of the spray dried powder low, as ?zo
PVP is hygroscopic.Germination ratesof eachformula- 5
tion detectedat the day of production were set as basic E
o
0
values.The lossof viability wasmonitored over a period c'
Tlme(mln)
of 2 months.
Fig. l. Survivalof Beauueriabrongniartii(A) and Metarhizütmanisop-
2.9. DeterminationoI the residualmoisturecontentof the &lae(B) heatedin a compositefluid of 5% (w/v) skim milk and 1.25%
(w/v)PVP K90 at 40'C (a), 45'C (D), 50'C (A), 55'C (l), and 60oC
formulations (O) for 0.5, l, 2, 4, 8, and l 5 min. The resultsarc meanst SD basedon
data from triplicate heat challengeexperimentsand were compared to
Thermogravimetricalanalysis(TGA) was performed the control. Untreated conidia of 8. brongniartii and M. anisopliaeof
by a thermogravimetricanalyzerTGAT (Perkin-Elmer; 95 and94%o viability havebeentaken as controls,respectively.
 A. Horaczek, H. VienrsteinI Biological Control 3I (2004) 65-71

resultedin a 17 and 43yoreduction of activity, respec- dia. B. brongniartüshowedgerminationratesof 6+.2%

tively. M. anisopliaetolerated an exposureto 50oC for
2min without loss of activity, whereasB. brongniartü
showed30% reduction in conidial vitality after I min at
50oC.Continuingthe exposureto 50oC for 2 min led to
viability rates of 4%. SubjectingM. anisopliaeand B.
brongniartüto 55oCfor 30s resultedin viability ratesof
97 and 0oÄ,respectively.
3.2. Spray-drying
and M. anisopliael5+2% comparedto 50 and 98o/"of
the control. An increaseof the inlet temperaturesto
100'C resultedin an almost completeloss of activity
and humidity levelsof 5.1%.B. brongniartüand M. ani-
sopliaeshowedgerminationratesof 3 and 4o/oafter pro-
cessingat 100'C inlet temperature,
respectively.
The processofspray-drying resultedin dust-free,free-
microcapsules.
flowing,and fast-releasing Due to SM the
conidia weredispersiblein aqueoustest media.

Table I illustratesthe influenceon the germinationof 3.3. Freeze-drying

various processparametersadjusted during spray-dry-
ing experiments.
The outlet air temperaturewas found to be the key
parameterto be controlled. It is a function of inlet air
temperature,the deliveryrate of sampledispersionsand
the aspirator capacity. As the aspirator capacity was
fixedat 35m3h-1,the feedsprayratewasvariedfrom 1.4
to 3mlmin-l dependingon the adjustedinlet tempera-
ture to keepthe outlet air and consequentialthe product
temperatureas low as possibleto ensureminimal heat
stressfor the fungal material.Condensationin the spray
dryer was avoided.Both fungal isolatesexpressthe same
germination after spray-drying. An exposure to 60/
40+2'C inlet/outlet temperaturesalready resultedin a
severedeclineof 65t2oÄ in conidial viability and mois-
ture contentsof 5.7Yo.B. brongniartii and M. anisopliae
respondto spray-dryingat 60/40* 2 oC inlet/outlet tem-
peratureswith survival ratesof 37 and 35olo,respectively.
An increaseof 20oC of the inlet temperaturedid not
alter the vitality as B. brongniartü and M. anisopline
showedsurvival rates of 35 and 34Yoand moisturecon-
tents of 5.4Yo.Comparing germination rates of freshly
harvestedand processedconidia after l6h incubation
indicateda severedelayin the germinationof dried coni-
By dividing the processof freeze-dryinginto its two
components,freezing and drying, it was possible to
study the effectsofboth processes on fungal viability.
Fig.2 shows that B. brongniartii and M. anisopliae
survivedthe rigors of freezingto -80oC and subsequent
thawing to ambient temperaturesdispersedin the sus-
pending media without loss of viability as germination
rates of 9l and 92Yowereachieved.The resultsindicate
that the processof drying was responsiblefor killing 30
and 9lYo of B. brongniartü and M. anisopliaeconidia,
respectively.Theseresultsclearly demonstratethe differ-
ing requirementsfor speciesto surviveprocessing.Fig. 2
clearly shows the lyoprotective effect of the composite
suspendingmedia for B. brongniartii.Germinationrates
of 68Y"wereachievedbut a delayin germinationbecame
apparent. After l6h incubation, only 3o/oof the pro-
cessedconidia showed germination compared to 50oÄ
germinationof freshlyharvestedconidia.For M. anisop-
liae the suspendingmedium was unable to provide pro-
tection during the drying step. The lyophilization
processresultedin moisturelevelsof 4.3%.The lyophiliz-
ateswere dust-free,provided a sufficientphysicalstruc-
ture and wereeasyto reconstitutein aqueousmedia.

Table I
Processparametersadjusted for spray-drying and the resulting survival of Beanseriabrongniartii and Metarhizium anisopliae
air, 700Nl h-t
Spray-nozzlediameteq0.7mm; aspirator,35m3h-l; flow of compressed
oC
60 Inlet temperature 80oC Inlet temperature 100'C Inlet temperature
Germination ra'te(Y")after l6h 51.5
r 6.2 I 1.3+ 6.1 9.4+3.2 0
incubation of B. brongaiartii
Germination r:;te (%o)after ?Ah 96.4+ 1.5 36.7+6.9 35.0+ 7.38 3.0+ 2.1
incubationof B. brongniartü
Germinationrate (%) after 16b 93.2+7.3 17.4+2.5 15.6
+ 5.4. 0
incubation of M. anßopliae
Germination rate (Vo)after 24h 96.1
+2.5 33.7*2.5 34.2+8.3 3.8+ 3.7
incubation of M. anisopliae
Outlet ('C) 40*.2 53+2 4 t+ 2
Product (oC) 30+,2 35+.2 4 tt 2
Suspensionfeedtrnt min-t) t.4 2.3 5

Moisture content (7o) 5.7 5.4 5.1

Processtime (min) l8 ll 8
Germinationratesweredetectedafter 16 and 24h. The resultsare means4 SD basedon data from triplicate spray-dryingexperiments.Freshly
harvesteduntreated conidia servedas controls.
 A. Horaczek. H. Viernstein / Biological Control 3l (2004 ) 65-71
69

A
s'100
.s 80
9oo
E
540
.E',o
$ o
(,o
tr 16 h incubation 824 h incubation

B
F.100
.E 80
*oo
5+o
.E zo
E
o
o
(,
g 16 h incubation @ 24 h incubation

Fig' 2 Survival of Beauaeria brongniartii (A) and Metarhiziturt anisopliae (B) subjected to freezing
and thawing (l) and lyophilization dispersed in
0'l% (wlv) Tween 80 solution (2) and in the composite carrier matrix system (3). Germination rates were
detected after l6 and 24h.The results are
means A SD based on data from triplicate experiments. Freshly harvested untreated conidia of B.
brongniartii and M. anisopliae of 95 and 94u/ovia-
bility served as control, respectively.

3.4. Fluid-beddrying Granules resulting from fluid-bed drying were dust-

Fluid-beddrying experimentswereperformedat the
same inlet/outlet temperature regimes adjusted for
spray-drying.Processingat an inlet temperatureof
100"C was omitted as insufficientviability rates were
achievedby spray-drying.The PVp proporrion was
increasedto 5Yo(w/v) to increasethe binding power,
resultingin granule formulations.In Table 2 various
processparametersand the resultingeffectson conidial
vitality aresummarized.
Beauueriabrongniartii and M. anisopliaeshowedger-
mination ratesof 16 and 4o/oafter processingat 60oC.
An inlet temperatureof 80"C resultedin conidial sur-
vival of 9 and 5nÄ.
free, free-flowing,and had moisture levelsof 5,,1,.
3.5. Determination of storage stability
All three drying technologies,investigatedduring this
study, resulted in formulations of moisture conrents
<6"Ä. The addition of silica gel capsulesto spray dried
powders ensured humidity levels of <5oÄ, as Moore
et af. (1996) recommendedmoisture contents of 4-5%
for prolonged conidia storage.Storage at increasedtem-
peratures acceleratedthe loss of viability. Table 3 sum-
marizes the results of storage tests for both species.The
low survival levelsafter fluid-bed drying were reflectedin
the complete loss of viability within 2 weeks storage at

Table2
Processparameters adjusted for fluid-bed drying and the resulting survival of Beauueria brongniartii
and Metarhiziturt anisopliae
Spray-nozzlediameter,0.8mm; spraygas,dried air; spraygaspressure,2 atm

Control 60'C Inlet temperature 80'C Inlet temDerature

Germination rate (Vo)after l6 h incubation of B. brongniartii 5l+3 4.8+ 2.5 5 . 3+ 2 . 7
Germination rate (o/o)after 24h incubation of B. brongniartii 9 6 +I 16.3 +2.4 8.8+ 5.2
Germination rate(oÄ) after l6h incubation of M. anisopliae 93+3 2.5+ 2.1 2.3+ t.0
Germination rate (oÄl after 24h incubation ol M. anisipliae 9 6+ 2 12.3+ 4.4 4.8+2.2
Outlet ("C) 3 7+ 3 42+2
Product ("C) 3 7+ 3 4 t+ 2
Spraying time (min) 6 o
Moisture content (7o) 5.0 5.1
Germination rates were detected after l6 and 24h. The results are means * SD based on data from
triplicate drying experiments. Freshly-hlr--
vesteduntreated conidia servedas controls.
 A. Horaczek, H. ViernsteinI Biological Control 3I (2004) 65-71

Table 3
Shelflife of formulatedconidia of both isolatesexposedto differentstoragetemperatures
Formulation

60c r00(r0) r00(+0) 94.5(r7.8) 83.0(+5.7) 8r.5(+3.5) 78.0(fr.4)

20'c r00(*0) 71.5(*2.r) 1.3)
58.0(+r 33.0(+21.2) 2.s(+3.5) 0
30'c 62.0(+s.7) 34O(+2.8) r2.s(+7.8) r.5(+2.1) 0 0
60c 54.0(+s.7) 51.0(r5.7) 49.0(r2.8) 42.5(+3.5) 35.5(r3.5) 28.0(+1.4)
20"c 36.0(+7.8) 3r.5(+7.8) 25.0(+2.8) 18.0(+2.8) 14.5(*2.r) 0
30'c 28.0(+1.4) 20.0(+4.2) 6.s(r9.2) 6.s(+9.2) 0 0
NI 60c 62.5(r5.0) 53.5(+17.7) 52.0(a15.6) 46.0(+12.7) 39.0(r2.8) 34.5(+3.5)
20.c 47.0(+8.5) 48.0(rr5.6) 28.0(+8.5) l r.5(+6.4) 8.0(arr.3) 5.5(+7.8)
30'c 37.0(*
| 5.6) 2s.5(+5.0) 9.0(r12.7) 2.0(r2.8) 0 0
The means* SD of five samplesstoredat the specifiedtime and temperatureis givenin the table.(I) lyophilizatesof Beauueriabrongniartii,(ll)
spraydried powdersof B. brongniartir,and (III) lyophilizatesof Metarhiziumanisopliae.

l0oC for both species.Therefore,thesedata are not
shownin the table.
In spray dried powders, germination rates were
reduc€dby 50%after2 monthsstorageat l0oC. At 20oC
a steadydeclineof activity wasobservedresultingin via-
bility levelsof 6 and U/o after 2 months. At 30oC both
speciesshowedgermination rates of 0oÄafter 7 weeks.
The lyophilizateswere found to be the most stablefor-
mulation for B. brongniartii.Storagetestsresultedin sur-
vival rates of 78/o stored at l0oc for 2 months.
Completelossof activity occurredafter 7 and 8 weeksat
30 and 20oC, respectively.No storage tests were per-
formed for M. anisopliaelyophilizatesas survival rates
of 4oÄresultedafter processing.
4. Discussion
Limited desiccationand temperaturetoleranceare the
most important constraintsfor processingentomopatho-
genicfungi by commonlyuseddrying methods(Daemen
and van der Stege,1982).For spray-and fluid-beddrying,
the time of drying is very short in comparisonto other
drying methods.During our experimentsprocesstimes
<20min wererequired.Bartlettand Jaronski(1988)and
Bailey and Rath (1994)considerthat rapid drying of M.
anisoplineconidiaat < 30-35oCis critical to preservation
of conidia viability. For comparison,high germination
rateshad beenachievedwith atmospherictray drying-a
processby which organismsand carriersare slowly air-
dried for l6h or longer. High viability and longevity
levelshad been reported (Chen et al., 1990;Feng et al.,
1994;Hong et al., 2000;Jenkinset al., 1998;Shi, 1988).
Freeze-drying,a 24h lasting drying process,is the most
convenientand successfulmethod for preservingmicro-
organisms.However,not all strains survive the process
and amongthosesurviving,quantitativeviability ratesas
low as 0.1%havebeenreported(Atkin et al., 1949;Smith
and Onions, 1983). Many authors have reported the
positive effectsof SM as a suspendingagent prior to
freeze-drying(Morichi and [rie, 1973;Sinhaet al.,1974).
Fargueset al. (1979) demonstratedthat lyophilization
was su@essfullyusedby mixing blastosporesof B. bas-
smc (Balsamo) Vuillemin with SM and glycerol. The
resultsof germinationtestsshowthat lyophilizationwith
a SM/PVP matrix is a promising drying method for
B. brongniarlliwhereasthe processresultedin complete
loss of viability for M. anisopliae.Theseresultsunder-
score that the formulation processhas to be designed
carefully for the particular requirementsand limitations
of each particular organism (Rhodes, 1993).When the
germinationspeedof untreatedand dried conidiais com-
pared,the prolongedgerminationtime showsthe strong
influenceof drying on conidia activity. Injuries to cell
membranesand proteins might be responsiblefor the
prolonged germination (Carpenter and Crowe, 1989;
Dong et al., 1995;Prestrelski et al., 1993).
From an industrial point of view,there are increasing
needsto predict and control the stability of dried ento-
mopathogenic fungi because their use is constantly
growing due to their environmentalcompatibility com-
pared to traditional fungicides.Further investigations
will show if the addition of disaccharidesto the SM/PVP
matrix will havea positiveinfluenceon conidial survival
during spray- and fluid-bed drying. Stephanand Zim-
mennann (1998),for example,showedviability levelsof
90oÄfor submergedconidia of entomopathogenicfungi
by spray-dryingin a SM matrix enrichedwith sugarbeet
syrup.Lyophilization resultsin promisingviability levels
for B. brongniartübut the longevity of lyophilized coni-
dia at higher temperatureshas to be improved. There-
fore, further experiments should demonstrate if
protectantswith higher glasstransition temperatureval-
ueswill improvethe shelflife.For M. anisopliae,lyophi-
lization in the SM/PVP suspendingmedia resulted in
completeloss of viability, showing the need for further
investigationsof alternative protectants.According to
Font de Valdez et al. (1983),protection afforded by a
given additive during dehydration will vary with the
speciesof microorganism.
 A. Horaczek, H. Viernstein I Biological Contol 3l (2004) 65-71
Spray application of dry aerial entomopathogenic
fungal conidia is difficult due to their hydrophobicity.
This characteristicrenderstechnicalpowdersextremely
dustyand difficultto suspendin water.All threetypesof
produced formulationseliminatethe dust hazardsby
embeddingthe conidia in a hydrophiliccarrier matrix,
makingthemeasilydispersible in the sprayliquid.
Acknowledgment
We thank F. Joh. Kwizda GmbH for the opportunity
to participatein the EU-projectBIPESCO(FAIR6-CT-
4105).
References
Ashwood-Smith,M.J., Warby, C' 1972.protectiveeffect of low and
high molecularweightcompoundson the stability of catalasesub_
jectedto freezingand thawing.Cryobiology9, 137_140.
Atkin, L., Moses,W., Gray, P.p., 1949.The preservationof yeastcul-
ture by lyophilization.J. Bacteriol.57.575-S'lg.
Bailey, L.A., Rath, A.C., 1994.production of Metarhizfun anisopliae
sporesusing nutrient-impregnatedmembranesand its economic
analysis.Biocontrol Sci.Technol.4, 297-?l07.
Bartlett, M.C., Jaronski,S.T.,1988.Massproduction of entomogenous
fungi for biologicalcontrol of insects.In: Burge,R.N. (Ed.), Fungi
in Biological Control Systems.ManchesterUniversity press,New
York, pp. 6l-85.
Bradley,C.A., Black, W.E., Kearns,R., Wood, p., lgg2. Role of pro_
duction technologyin mycoinsecticidedevelopment.In: Leatham,
G.F. (Ed.), Frontiers in Industrial Mycology. Chapmanand Hall,
New York, pp. 160-173.
Bühler,V., 2001,Kollidon: Polyoinylpyrrolidone for thepharmaceutical
industry.
Burges,H.D., 1998.Formulation of mycoinsecticides. In: Burges,H.D.
(Ed.),Formulation of Microbial Biopesticides:BeneficialMicroor_
ganisms,Nematodesand SeedTreatments.Kluwer Academicpub_
lishers,Dordrecht,pp. l3l-187.
Butt, T.M., Jackson,C., Magan, N., 2001.Introduction-Fungal bio-
logical control agents:progress,problemsand potential. In: Butt,
T.M., Jackson,C., Magan, N. (Eds.),Fungi as Biocontrol Agents:
Progress,Problemsand Potential.CABI publishinC,pp. l_g.
Carpenter,J.F.,Crowe,J.H., 1989.An infrared spectroscopicstudy of
the interactionsofcarbohydrateswith dried proteins.Biochemistry
28.391G3922.
Chen,C.L.,Wu, J.W.,Li,Z.Z,Wang,Z.X.,Li, y.W., Chang,S.H.,yin,
F.M., Wang, X.P., Dai, L.Y., Tao, L., Zhang,y.A., Tang, J., Ding,
G.G.,Gao, Z.H.,Tan, Y.C., 1990.Appticationof microbialpesti_
cidesin IPM. In: Chen,C.J.(Ed.), IntegratedManagementof pine
Caterpillarsin China. China Forestry publishing House, Beijing,
pp.214-308.
Daemen,A.L.H., van der Stege,H.J., 1982.The destructionof enzymes
and bacteriaduring the spraydrying ofmilk and whey.2.The effect
of the drying conditions.Neth. Milk Dairy J. 36,2ll-229.
7l
Dong,A., Prestrelski, S.J.,Allison,S.D.,Carpenter,J.F.,1995.Infrared
spectroscopicstudiesof lyophilization- and temperature_induced
protein aggregation.J. Pharm.Sc| 84,415424.
Fargues,J., Robert,P.H.,Reisinger,O., 1979.Formulation desproduc_
tions de massede l'hyphomycöteentomopathogöneBeauueriaen
vue des applicationsphytosanitaires. Ann. Zool. Ecol. Anim. ll,
247_257.
Feng,M.G.,Poprawski,T.J.,Khachatourians, G.G., 1994.production,
formulation and application of the entomopathogenicfungus
Beauueriabassianafor insectcontrol: current status.Biocontrol Sci.
Technol.4.3-34.
Ferron, P., 1981.Pestcontrol by the fungi BeauueriaandMetarhizium.
In: Burges,H.D. (Ed.), Microbial Control of pestsand plant Dis_
eases1970-1980.AcademicPress,New york, pp. 465492.
Font de Valdez,G., de Giori, G.S., Ruiz Holgado, A.p., Oliver, G..
1983.Comparativestudyofthe efficiency ofadditivesin protecting
lactic acid bacteriaagainstfreezedrying.Cryobiology20, 560_566.
Hieda, K., Ito, T., 1973.Freeze-dryingof biological materials.In: pro-
ceedingsofC-l Symposium,Sapporo.paris: InternationalInstitute
of Refrigeration.
Hong, T.D., Jenkins,N.E., Etlis, R.H., 2000.The eflectsof duration of
developmentand drying regime on the longevity of conidia of
Metarhizitunflauouiride.Mycol. Res.104,662465.
Jenkins,N.E.,Heviefo,C., Langewald,J.,Cherry,A.J.,Lomer,C.J.,199g.
Developmentof massproduction technologyfor aerialconidia for
useasmycopesticide. BiocontrolNewsInform. l9,2lN_31N.
Moore, D., Douro-Kpindou,O.K., Jenkins,N.E., Lomer, C.J.,1996.
Effectsof moisture content and temperatureon storageof Meta-
rhiziumflaoouirideconidia.Mycol. Res.6, 5|_6L
Morichi, T., Irie, R., 1973.Factorsaflectingrepair of sublethalinjury in
frozenor freeze-driedbacteria.Cryobiology I 0, 393_399.
Morris, G.J.,Farrant,1., l9T2.Interactionsof cooling rate and protec-
tive additive on the survival of washedhuman erythrocytesfrozen
to - 196.Cryobiology9, l 73-l 8 l.
Nath, S.,Satpathy,G.R., 1998.A systematicapproachfor investigation
of spraydrying processes. Drying Techn. 16,ll73_llg1
Prestrelski,S.J.,Tedeschi,N., Arakawa,T., Carpenter,J.F., 1993.Dehy_
dration-inducedconformational transitions in proteins and their
inhibition by stabilizers.Biophys.J. 65,66147 l.
Rhodes, D.J., 1993.Formutations of biologicat control agenrs.In:
Jones,D.G. (Ed.), Exploitation of Microorganisms.Chapmanand
Hall, London,pp.4l l-439.
Shi,2.M., 1988.Technologyfor conidial preparationof Beauueriabas-
siana.In: Li, Y., Li, 2., Liang,2.e., Wu, J.W., Wu, 2.K., Xu, e.F.
(Eds.),Study and Application of EntomogenousFungi in China,
vol. l. AcademicPeriodicalPress,Beijing,pp. l l4-l 15.
Sinha,R.N., Dudani, A.T., Ranganathan,8., 1924.protectiveeffectof
fortified skim milk as suspendingmedium for freeze-dryingof
differentlactic acid bacteria.J. Food Sci. 39,641-642.
Smith, D., Onions,A.H.S.,1983.The preservationand maintenanceof
living fungi. CommonwealthMycologicallnstitute, Kew, England,
p.51.
Stephan,D., Zimmermann,G., 1998.Developmentof a spray_drying
techniquefor submergedsporesof entomopathogenicfungi. Bio_
control Sci.Technol.8. 3-l l.
Teixeira,P., Castro,H., Kirby, R., 1994.Inducible thermotolerancein
Lactobacillusbulgaricus.Lett. Appl. Microbiol. lg, 2lg_221.
Teixeira,P.,Castro,H., Kirby, R., I995.Spraydrying as a method for
preparingconcentratedculturesof Lactobacillusbulgaricus.J. App.
Bact.78,456462.