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Strain selection, production, and formulation of the biological

plant vitality enhancing agent FZB24

Bacillus subtilis
H. Junge, B. Krebs, M. Kilian
2 Strain selection and systematic
identification of FZB24

Bacillus
subtilis
So far 65 fully characterized species
have been assigned to the genus Bacillus.
With new diagnostic methods, it is now
possible to recognize greater genetic
and physiological diversity within indi-
vidual species. As a consequence, new
taxonomic groups could be seperated in
the future, and some species could be
assigned to different groups.
The Bacillus subtilis group includes e.g.
the species B. amyloliquefaciens, B. subtilis,
B. pumilus, B. licheniformis, and B. firmus,
which are characterized by acid forma-
tion from various sugars, including glu-
cose, under aerobic conditions, and which
have ellipsoid spores that are not larger
than the parent cell (Priest 1993).
FZB24

Bacillus subtilis, which had been

isolated from natural habitats in Bran-
denburg, Germany, was identified as
B. subtilis by the methods of Bergey
(1986) on the basis of its morphological,
biochemical, and serological propert-
ies (Juhr, 1992). The identification as
Bacillus subtilis has been confirmed by
computer-assisted diagnostic systems that
are also based on biochemical parame-
1 Development concept for biological
products
Bacillus subtilis, known as hay bacterium,
is a well known micro-organism, and
various strains are already used industri-
ally in diverse fields. The aim of this pro-
ject was to develop marketable products
from micro-organisms of the genus
Bacillus for use in biological and inte-
grated plant protection. The necessary
criteria for the successful development
and marketing of biological products
are: reliable effect, suitability for appli-
cation by conventional techniques, and
competitive treatment costs per ha for
both the farmer and the gardener. A
low-cost production method, constant
product quality, satisfactory storage
stability, and formulation capability are
indispensable too. FZB24

meets these
criteria, as explained below.
The strain selection process that led to
FZB24

took several years. Numerous

experiments had shown that FZB24

was the strain that reliably promoted

growth and yields and reduced losses
due to soil-borne pathogens in many
species of plants. In addition, this strain
also fullfilled all the requirements on the
production and formulation sides.
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ters, such as the VITEK

system (ano-
nymous, 1996) (Table 1). However, fatty
acid analysis (Hoffmann-Hergarten, 1996;
Verbarg, 1996) also showed correlations
with the fatty acid pattern of Paenibacillus
macerans and B. amyloliquefaciens.
The Bacillus subtilis strain FZB24

forms the typical wrinkled colonies of

Bacillus subtilis in surface cultures on
solid PD-agar (Fig. 1). Vegetative cells or
spores are found in liquid cultures, de-
pending on the age of the culture (Fig. 2).
Fig. 1: Colony forms of FZB24

Bacillus subtilis on solid PD agar after incubation for 5 days at 25C

(Photo: Dr. U. Steiner, University of Bonn)
Fig. 2: Vegetative cells and spores of FZB24

Bacillus subtilis
(Photo: Dr. U. Steiner, University of Bonn)
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3 FZB24

Bacillus subtilis produc-

tion process
FZB24

Bacillus subtilis is produced in

liquid cultures as indicated by the scheme
in Figure 3.
The starting cultures are prepared from
a lyophilized stock culture. The charac-
teristic properties of the strain are
checked on selected single colonies, and
only tested colonies are subsequently
cultivated. Spore solutions are prepared
from those colonies and frozen in por-
tions for the inoculation of precultures.
This procedure guarantees a uniform
strain identity and a uniform starting
material for fermentations over a period
of many years.
For a production run, a preculture is
prepared in a shaking flask from one of
the frozen cultures. After an adequate
cultivation time, this preculture is used
for the inoculation of a fermenter. The
preculture procedure is repeated several
times, in accordance with the intended
volume of the main fermentation; the
fermentation volume can be increased
in a ratio of 1:100 in each step.
The main culture takes place in a com-
plex culture medium partly based on
soya bean products. Natural starting
materials of this type are used only if
certificates confirm that they are free
from genetically modified constituents.
This is important for subsequent use in
organic farming. The natural nutrient
components are necessary to attain high
cell densities and hence high yields of
heat-resistant spores of B. subtilis. Com-
plex natural nutrients are a source for a
number of growth factors and trace
elements, mixtures of free amino acids
for the exponential growth of the pop-
ulation and for complex organic nitro-
gen sources (proteins, peptides). These
provide a continuous supply of the
necessary amino acids and small peptid-
es for the growth of the culture in the
transition phase. This continuous supply
results from the action of the strain-
specific extracellular proteases and
peptidases, the formation of which is
triggered by starvation in respect of
individual amino acids after the first
restriction of growth (end of the loga-
rithmic phase). In addition to the easily
utilizable nitrogen sources, the complex
nutrients also provide inorganic phos-
phate, which is released from these
nutrients by the sporulation-specific
alkaline phosphatase, and which controls
the sporulation process and the develop-
ment of the heat resistance of the spores.
The oxygen-content, the pH, etc. are
continuously monitored during the
fermentation. Examples of the typ-
ical growth curves obtained and the
changes in the medium are shown in
Figures 4 and 5.
The growth curve is characterized by a
logarithmic phase lasting about 4 hours
and a prolonged transition phase. The
maximum cell density of approximately
1.0 x 10
10
CFU/g, with > 95% sporulation
of the cells, is reached about 20 hours
after inoculation. The fermentation is
ended at this point.
The solid components of the culture
solution, i.e. the spores, cells, and nutri-
ent residues, are separated from the
liquid components and the metabolic
products formed in the course of the
fermentation. The volume is reduced to
about 1/10 of its initial value by aseptic
separation. The biomass obtained, which
is also known as bioslurry because of its
consistency, is then dried and formulated
by suitable processes.
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4 Drying and formulation of FZB24

Bacillus subtilis
Various processes have been tested
for the drying and formulation of the
spores of FZB24

Bacillus subtilis. Spray

drying, fluidized-bed drying, and freeze
drying are fundamentally suitable for
drying the B. subtilis spores, which are
resistant, and for formulating them with
the aid of additives. Temperatures of up
to 60C can be used intermittently in
the drying process without causing any
appreciable losses of viable spores.
Quartz sand was initially used as a car-
rier for the B. subtilis spores, and later
soluble fertilizer salts such as potassium
nitrate or ammonium sulphate. The gran-
ular products yielded by this formula-
tion procedures had spore contents of
> 5.0 x 10
10
CFU/g and residual moisture
contents of about 1%, but showed a
relatively wide grain size distribution.
The use of grain starch as a carrier of-
fered the advantage that since this is an
organic material, the product could then
also be used in organic farming, for
which even traces of fertilizer salts in the
product are prohibited. However, starch
as a carrier leads to a finer grain size, so
that only a very fine water-dispersible
granulate could be produced. The dis-
persibility of the formulations on starch
was also distinctly better than when
potassium nitrate was used as carrier. In
addition to its formulation character-
istics, the carrier material can also have a
certain influence on the biological
effect of the product. Conceivable
effects are improved adhesion of the
treatment suspension to the potato
tubers and a support for the bacterium
during its establishment in the rhizos-
phere as a consequence of the small sup-
ply of nutrients. A comparison of KNO
3
and maize starch as carrier materials
showed small but statistically non-veri-
fiable advantages for the organic mate-
rial (Fig. 6).
The residual moisture-content of the
product has a decisive influence on the
storage stability of the formulation. Par-
ticularly under unfavorable storage con-
ditions, if the moisture-content is more
than 18%, the spores may die relatively
quickly and the product may become
mouldy. The dependence of the storage
stability on the residual moisture-content
is illustrated in Table 2 for storage under
extreme conditions at 54C, which is
used to simulate prolonged storage.
In the case of products with starch as the
support, the residual moisture-content
can be reduced to less than 12% by post-
drying under vacuum.
Optimization of the spore-content of the
FZB24

formulations, together with low

residual moisture-content and diffusion-
tight packaging, guarantees that the con-
tents of viable spores in the product
remains sufficiently high for at least
2 years. This is confirmed by storage
tests at +54C, at normal room temper-
ature up to 30C, and at alternating tem-
peratures of 15 to +30C (Fig. 7).
A second FZB24

formulation with talc

as a substantially inert carrier has been
specially developed for use as a dry
treatment, e.g. for potatoes. The use of a
lower spore concentration allows direct
application of the undiluted product with-
out any impairment of the adhesion to
the surfaces of seeds and seed potatoes.
The result of these studies was the
characterization and development of the
Bacillus subtilis strain having the desi-
gnation FZB24

(Krebs et al. 1998). Nu-

merous trials had shown that FZB24

is
Pflanzenschutz-Nachrichten Bayer 1/00, 1
reliably effective in promoting plant
growth and in reducing losses due to
soil-borne pathogens. On the basis of
its mode of action growth promotion,
stress reduction, and resistance induc-
tion combined with weak antifungal
effects FZB24

Bacillus subtilis is
registered as a plant strengthening
agent with the German Biological In-
stitute for Agriculture and Forestry.
FZB24

has now also been registered in

the USA and in Austria, and registration
in other countries is currently being
prepared or expected.
5 Summary
The Bacillus subtilis strain FZB24

is the
result of years of study in which various
strains of Bacillus were selected on the
basis of biological effectivity and suita-
bility for production. FZB24

is produc-
ed in a multi-stage liquid fermentation
process from a stock culture that guar-
antees a uniform strain identity. The
spores formed in this process are separa-
ted from the culture broth and then
dried and formulated together with pro-
tective colloids, inert material, and other
additives. The formulated end product
has a storage stability of at least 2 years.
FZB24

Bacillus subtilis is registered in

Germany as a plant strengthening agent.
Preparations are being made for its regis-
tration in other countries.
Zusammenfassung
Stammselektion, Produktion und Formu-
lierung des biologischen Pflanzenstr-
kungsmittels FZB24

-Bacillus subtilis
Der Bacillus subtilis Stamm FZB24

ist
das Ergebnis langjhriger Studien, in
denen verschiedene Bacillus-Stmme
auf Wirkung und Produzierbarkeit hin
selektiert wurden. FZB24

wird ausge-
hend von einer Stammhaltung, die eine
gleichbleibende Stammidentitt gewhr-
leistet, in einem mehrstufigen Flssig-
fermentationsverfahren produziert. Die
dort gebildeten Sporen werden durch
Separation von der Kulturbrhe abge-
trennt und zusammen mit Schutzkoll-
oiden, Inertmaterial und weiteren Zu-
stzen getrocknet und formuliert. Das
formulierte Endprodukt besitzt eine
Lagerstabilitt von mindestens zwei
Jahren. FZB24

-Bacillus subtilis ist in

Deutschland als Pflanzenstrkungsmittel
registriert. Die Registrierung in weiteren
Lndern befindet sich in Vorbereitung.
Rsum
Slection des souches, prparation et
formulation de lagent biologique sti-
mulateur de vigueur vgtale FZB24

-
Bacillus subtilis
La souche FZB24

de Bacillus subtilis
est le rsultat de plusieurs annes dtudes
qui ont permis la slection de diffrentes
souches de Bacillus, en fonction de leur
efficacit et des possibilits de leur pro-
duction. Le FZB24

est produit par

un procd de fermentation en phase
liquide en plusieurs tapes, partir dune
souche mre qui garantit une identit
constante de la souche. Les spores qui
sy forment sont isoles par sparation
du bouillon de culture et sont sches et
formules avec des collodes protecteurs,
un matriau inerte et dautres additifs.
Le produit final formul a une stabilit
au stockage dau moins 2 ans. Le FZB24

-Bacillus subtilis est homologu en

Allemagne en tant quagent stimulateur
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de vigueur vgtale. Lhomologation dans
dautres pays est en cours de prparation.
Resumen
Seleccin de cepa, preparacin y formu-
lacin del vigorizante biolgico FZB24

-
Bacillus subtilis
La cepa FZB24

de Bacillus subtilis es el
resultado de estudios durante aos, en
los que se han seleccionado las mejores
cepas de Bacillus en cuanto a eficacia
y producibilidad. FZB24

se produce
mediante un proceso de fermentacin
lquida de varias etapas, partiendo de
una cepa original que garantiza una
identidad constante. Las esporas for-
madas se separan del caldo de cultivo y
despus se secan y formulan junto con
coloides protectores, material inerte y
otros aditivos. El producto final for-
mulado posee una estabilidad al al-
macenaje de 2 aos como mnimo.
FZB24

-Bacillus subtilis est registrado

en Alemania como vigorizante para
vegetales. Se est tramitando el registro
en otros pases.
Posmuo
eequn m1auua, npusn1n
u npu1nneuue npenapa1unu
pum epennme u-
uue pe1na FZB24

-
Bacillus subtilis
L1aVV FZB24

a1epuu Bacillus subti-
lis nene1n pesb1a1V Vue1uux
ueea1ebux pa1, e xe
1pux npeuab eeun pas-
uuuux m1aVVe a1epuu e ane1e
se1ueu1u u esVxu1e
penpuu. Hxn us eu ep-
xauun m1aVVa, eneuueakux e
ueusVeuuk ueu1uuu1b, FZB24

npuseu1n n Vu1neuua1
xu1u epVeu1a1ueu 1exu-
uu. paseaemuen npu 1e-
nk1 n1eV enapauu 1 b1-
pabu nu1a1ebu peu, a sa1eV
ma1 eVe1e sau1uuVu -
uaVu, uuep1uuV Va1epuaV u pu-
Vu aeaVu u npu1aeueak1
npenapa1ueuk pV. p xpa-
ueuun ueuu np1a e pena-
pa1ueu pVe 1aene1 ue Veuee
2 e1. B epVauuu npenapa1 FZB24

Bacillus subtilis sapeu1pupeau a

epennkee pe1e n
pa1euu, e peu1paun e pux
1pauax uaxu1n e 1auu n-
1eu.
6 References
Anonymous 1996:
Instructions for use, bioMerieux Vitek, Inc.; 595 Ang-
lum Drive, Hazelwood Missouri 63042-2395, USA
Bergeys Manual of Systematic Bacteriology,
Vol. 2, Ed. Sneath, P.H.A.; Mair, M.E.; Holt, I.G.;
William & Wilkins, 1986, Baltimore
Hoffmann-Hergarten, 1996 personal communication
Juhr 1992:
FZB Biotechnik internal report
Krebs, B; Hding, B., Kbart, S., Alemayehu, M.,
Junge, H., Schmiedeknecht, G., Grosch, R.,
Bochow, H., Hevesi, M. (1998):
Use of Bacillus subtilis as biocontrol agent. 1. Ac-
tivities and characterization of Bacillus subtilis strains.
Z. Pflanzenkrankh. Pflanzenschutz 105, 181197
Priest F.G. (1993):
Systematics and Ecology of Bacillus. In: Sonens-
heine, A.L., Koch, J.A., Losick, R. (eds.). Bacillus
subtilis and other gram-positive bacteria: bioche-
mistry, physiology and molecular genetics. Ameri-
can Society of Microbiology, Washington
Verbarg, S. (1996):
personal communication
Pflanzenschutz-Nachrichten Bayer 1/00, 1
Table 1: Biochemical properties of FZB24

Bacillus subtilis determined on the ba-

sis of various identification cards of the Vitek

in-vitro diagnosis system.

Positive reaction denotes tolerance to or transformation of the test substance. For
details see instructions for use (anonymous 1996).
Reaction pattern on BACILLUS identification card
Sucrose + Trehalose +
Tetrazolium Red Palatinose +/
Tagatose Sorbitol +
Glucose + N-Acetyl-D-glucosamine +
Inositol + Amylopectin +
Galactose +/ Potassium thiocyanate +
Arabinose +/ 7% NaCl +
Xylose +/ Mandelic acid +
Mannitol + Oleandomycin
Raffinose +/ Sodium acetate
Salicin + Arabitol
Amygdalin +/ Polyamidohygrostreptin +
Inulin + Nalidixic acid
Ribose + Aesculin +
Maltose +
Additional reaction pattern on the GRAM-POSITIVE identification card
Peptone + Raffinose
Bacitracin + Salicin +
Ethylhydrocupreine hydrochloride + Sorbitol +
Hemicellulase +/ Sucrose +
6% NaCl + Trehalose +
10% bile + Arabinose
40% bile Pyruvic acid
Aesculin + Pullulan
Arginine monohydrochloride Inulin
Urea Melibiose
2,3,5-Triphenyltetrazolium Melezitose
chloride
Novobiocin sodium salt Cellobiose +
Dextrose + Ribose
Lactose Xylose
Mannitol + Catalase
100
7 Appendix
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Table 2: Influence of the residual moisture-content of the product on the storage
stability of Bacillus subtilis, illustrated by the results for three trial batches with
swellable starch as carrier material
Moisture-content 5% 11% 18%
of the product
Content of viable 100% 62% 29%
spores after storage
for 6 weeks at 54C
(5% sample = 100%)
Fig. 3: Scheme of the FZB24

Bacillus subtilis fermentation process

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Fig. 4: Typical development of the numbers of vegetative cells and spores of FZB24

during
the fermentation process
Fig. 5: Typical development of the pH value, lactose, ammonium and phosphate-content of the
fermentation broth during the fermantation process of FZB24

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Fig. 6: Influence of the two carrier materials of the FZB24

formulations on the yield of

potatoes after seed tuber treatment (Trials University of Bonn, Dr. U. Steiner 1996)
Fig. 7: Storage stability of FZB24

under different temperature conditions

Pflanzenschutz-Nachrichten Bayer 1/00, 1
Manuscript received: 14.6.00 Dr. Helmut Junge
Dr. Birgit Krebs
FZB Biotechnik GmbH
Glienicker Weg 185
D-12489 Berlin
Tel.: 030-67 057 0
Fax: 030-67 057 233
email: [email protected]
Dr. Michael Kilian
Bayer AG
Landwirtschaftszentrum Monheim
Geschftsbereich Pflanzenschutz/
Forschung
D-51368 Leverkusen
Tel.: 02173-383210 (Dr.Kilian)
email: [email protected]
104