Production and Utilization of Bacillus

thuringiensis

T. L. COUCH and D. A. ROSS, Chemical and Agricultural

Products Division, Abbott Laboratories, North Chicago, Illinois
60064

Summary
Biological insecticide formulations based on the entomogenous bacterium, Bacillus
thuringiensis, have begun to be used widely in agriculture. This spore-forming ba-
cillus can be grown in submerged culture and formulated to provide stable agricul-
tural formulations compatible with aerial and ground application systems. The safety
of these products to nontarget organisms and man is responsible for the growing
interest in their use on a variety of crops including pastures and forests. N o longer
a laboratory curiosity, B. thuringiensis is an economic alternative t o chemical insec-
ticides.

INTRODUCTION

In the past 15 years a subtle shift in the use of pesticides has

occurred in agriculture. There has developed at the University,
Federal, State, and grower levels an awareness that viable alter-
natives to the use of chemical insecticides exist. These alternatives
include specific narrow-spectrum chemicals, pheromones, and en-
tomopathogens that include viruses, bacteria, and fungi. These so-
called biorational insecticides are unique because they have a nar-
row spectrum and low or no toxicity to nontarget vertebrates and
invertebrates. They also are generally naturally occurring.
The purpose of this paper will be to describe the production and
utilization of one of these biorationals, Bacillus thuringiensis.

Bacillus thuringiensis

Description
Bacillus thuringiensis is a widely distributed, rod-shaped, spore-
forming, aerobic, gram-positive microorganism closely related to
Bacillus cereus. It is unique because it produces one or more par-

Biotechnology and Bioengineering, Vol. XXII, 4.1297- I304 (1980)

0 1980 John Wiley & Sons, Inc. OOO6-3592/80/0022-1297$01.OO
1298 COUCH AND ROSS

asporal crystals during the sporulation cycle and is pathogenic to

lepidopterous larvae. The most commonly cited classification sys-
tem for the varieties of B . thuringiensis is based on the antigenic
properties of the flagellar proteins. Fourteen major serotypes have
been described by Dr. de Barjac of the Pasteur Institute. Current
commercial strains have been identified as var. kurstaki, serotypes
3a and 3b. Both B. cereus and B. thuringiensis are common inhabi-
tants of the environment and grow in different soils. However, B.
thuringiensis is rarely isolated except from diseased insect larvae.

Historical
The earliest recorded isolation of a spore-forming bacteria from
insects was in Japan by Ishiwata in 1902. The insect was the silk-
worm and the organism isolated was the “sotto disease bacillus”.’
This organism was later described as Bacillus thuringiensis var.
sotto.
In 1915, Berliner isolated the type species of the group, Bacilfus
thuringiensis var. thuringiensis, from the Mediterranean flour moth,
Anagasta kuehniella.’ He described the disease in detail.
Between 1915 and 1950 other researchers experimented with
practical applications of B . thuringiensis against infestations of sus-
ceptible insects. They met with limited success. S t e i n h a d provided
the first report of a successful use of B . thuringlensis in the United
States (US) against the alfalfa caterpillar. However, commercial
enthusiasm for development of the bacterium was still nonexistent.
In review of the literature, the lack of any dramatic data on the
successful field use of the organism was probably due to early
ignorance of the mode of action behind the etiology of the disease.

Mode of Action
Hannay3r4described and named the diamond-shaped crystals as-
sociated with sporulating cultures of B . thuringiensis, parasporal
bodies. He speculated on their function in the mechanism of the
disease. Angus5-? conclusively demonstrated the toxicity of these
parasporal crystals to larvae of the silkworm.
Heimpel and Angus’ and Cookseys reviewed in detail the effect
of these protein crystals, also referred to as the 6-endotoxin, on
susceptible lepidopterous larvae. What is obvious from review of
these articles is that there is no simple description of the mode of
action. Basically, the proteinaceous toxin is hydrolyzed by the
combined enzyme activity of the digestive process and the alkaline
 PRODUCTION AND UTILIZATION OF B. THURZNGZENSZS 1299

pH environment of the midgut. The subunits of the crystal attack

the lining of the midgut causing paralysis via leakage of the alkaline
gut contents into the hemocoel.
The leakage from the lesions may be severe enough to kill the
larvae or cause changes favoring the growth and proliferation of the
organism resulting in septicemia. The combination of gut paralysis,
phago-depression, and septicemia results in larval death, usually
within 24-96 hr of ingestion of the parasporal crystals and endo-
spores. The development and intensity of the toxic septicemia pro-
cesses are extremely variable, depending both on the insect species
and strain of B. thuringiensis i n v ~ l v e d . ~
An additional toxin has also been described from cultures of B .
thuringiensis var. thuringiensis. Designated the p-exotoxin, it is
thermostable, affects orders of insects outside of that of the heat-
labile &-endotoxin,and has shown some mammalian toxicity. Be-
cause current manufacturers use strains that do not produce p-
exotoxin or must demonstrate its absence in the product, we will
not discuss its activity.
Basically elucidation of the mode of action helped commerciali-
zation by providing manufacturers with a recognized component,
the quantity and availability of which needed to be optimized to
provide an active product capable of performing under field con-
ditions.
Before describing the steps leading to the commercialization of
B. thuringiensis, it is worthwhile to review why much attention is
being given to biorational insecticides such as B. thuringiensis. This
attention is based on their selectivity and safety.

Selectivity and Safety

Bacillus thuringiensis is used primarily against Lepidoptera but
some recent new strains, primarily var. israelensis, has shown ac-
tivity against mosquito and blackfly larvae. Krieg'O compiled a list
of over 100 different species of Lepidoptera susceptible to B. thu-
ringiensis. Other insect orders are apparently unaffected by the
bacterium. No adverse affects have been recorded against other
insect or life forms such as man, pets, birds, fish, earthworms,
beneficial insects, or plants.'*l'
There has been no evidence of acute or chronic toxicity in rats,
guinea pigs, dogs, mules, swine, or other mammalian test animals.
Bacillus thuringiensis as a commercial formulation is self-limiting
in the environment. This is due to its failure to compete effectively'
with other soil bacteria, adverse growing conditions on the surface
1300 COUCH AND ROSS

of plants, and its susceptibility to weathering and solar

radiation.1*12~13
Because of this, B . thuringiensis has been granted an exemption
from tolerance requirements on all raw agricultural commodities
when applied to growing crops or after harvest when used in ac-
cordance with good agronomic practices.

UTILIZATION

Production
Since the early 1950s B. thuringiensis products have continued to
be commercially available in the US. These products all result from
deep-tank fermentation although various recovery techniques and
formulations are employed. One of the keys to successful commer-
cialization has been fermentation medium development. Most media
now employed used complete natural products as sources of carbon,
nitrogen, and trace minerals. The list of nitrogen sources used
include: fishmeals, cottonseed flour, corn steep liquor, soybeans,
autolyzed yeast, and casein.14 Carbohydrate sources used include:
hydrolyzed corn products, starch, and dextrose. l5 An important
aspect of B . thuringiensis preparations is that a significant part of
the final product consists of unused medial components and cell
debris. In addition, it has been shown that the toxic activity of a
given strain can vary significantly in different media. 15*16
The operating conditions used in large-scale deep-tank fermentors
are described in Refs. 15, 17, and 18. Fermentors up to 12000 gal
in size have been used for production.'' Initial pH in the medium
is typically adjusted to approximately 7.2 to 7.6. The amount of
inoculum can vary; however, the range of 2 to 5% of the total
fermentor volume is commonly cited in the literature. The Megna
patent6 also indicates a temperature of 3VC, a back pressure of 5
psig, and aeration at 5.3 ft/min superficial air velocity as being
suited for the cultivation of B . thuringiensis. The fermentation cycle
lengths mentioned in the literature vary between 14 and 72 hr; this
will depend on how rapidly sporulation is completed in a given
medium.
A variety of recovery techniques are employed for the commer-
cial production of B . thuringiensis. The recovery of the solids,
which contain the spores and &endotoxin, can be accomplished by
either centrifugation or filtration of the whole culture. 18319Cell pastes
obtained in this manner are further processed to obtain one of the
 PRODUCTION AND UTILIZATION OF B . THURINGIENSIS 1301

common product forms for B. thuringiensis insecticides: a dust,

wettable powder, or liquid.
Powders containing B. thuringiensis have been manufactured by
spray drying the whole culture or the cell paste." The cell paste
has also been dried in forced circulation or vacuum driers at 40-
50°C.20Powders obtained in this manner are then ground or com-
minuted and sifted to produce a finished powder.19Another method
that yields a powder is described in a Dutch patent.21In that process
the cell paste is washed several times with distilled water and then
freeze dried.
To obtain a liquid product, the process described by Briggs et
al.'O could be used. Their invention calls for the pH adjustment of
the cell paste (to about pH 3.5 to 4.5) and the addition of metal salts
and of oleaginous liquids to form a stabilized liquid preparation of
B. thuringiensis. Briggs also refers to another method of preparing
an oil-water suspension of B. thuringiensis. This was done by ball
milling a powder in admixture with xylene or diesel oils. The re-
sulting mixture was then emulsified with water and a small portion
of an emulsifying agent.

Formulations
During the 1950s and 1960s companies involved in the production
of B. thuringiensis used serotype I, B. thuringiensis var. thuringien-
sis as their standard organism. Four major brands of B. thuringiensis
var. thuringiensis were available (Table I).
Descriptions of early tests with these products can be found in a
review by Hall.'' This review includes an account of European
testing of similar products including Bactospeine, Biosper, Ento-
bacterin, and Sporeine. Ignoffo and Anderson" provide a more
complete listing of commercial products manufactured worldwide
using B. thuringiensis as the active ingredient.
The efficacy of early products was satisfactory against a number

TABLE I
Early Brands of B . thuringiensis Available as Commercial Products
Brand name Formulation Producer
Bakthane L-69 wettable powder and dust Rohm & Haas
Biotrol BTB wettable powder and dust Nutrilite Products
Parasporin wettable powder and dust Grain Processing Inc.
Thuricide wettable powder and dust International Mineral and Chemical
Corporation
1302 COUCH AND ROSS

of agricultural pests including: imported cabbage worm, Pieris

rapae ; diamondback moth, Plutella xylostelln; cabbage looper, Tri-
choplusia ni; and alfalfa caterpillar, Colias eurytheme. However,
the researchers and users of these products noted that efficacy was
dependent on both the biological and physical properties of the
formulations.
For example, on Cole crops, broccoli, cabbage, etc., results were
generally favorable with dusts but not with wettable powders. Cov-
erage was more uniform when dusts were applied and was easier to
regulate; coverage with the wettable powders was variable, since
these often were difficult to mix and when suspended, plugged the
nozzle screen and spray valves.z3Many harsh judgments by the end
user were directed against B . thuringiensis instead of the particular
formulation.
During the early years, insect pathologists such as Jac-
ques and and Tanada,26began experimentation with spreader
stickers to increase the efficacy of B . thuringiensis sprays in crop
systems. Lewis and Conno1az7assessed the effects of nonvolatile
carriers on application efficiency against the gypsy moth. However,
consistent predictable results against the range of susceptible insects
were never achieved with the early formulated products.
The number of manufacturers of B . thuringiensis products de-
creased considerably in the mid-1960s. Of the two remaining, In-
ternational Minerals and Chemicals, Inc. produced a flowable Thur-
icide which facilitated mixing and application. Biotrol and
Thuricide, the two remaining B . thuringiensis products, received a
federal label and acquired a small percentage of the insecticide
market. There use was generally restricted to application as clean-
up sprays on lettuce and Cole crops for loopers, cabbage worms,
etc., just prior to harvest. Because B . thuringiensis was exempt
from tolerance requirements, these materials provided the farmer
with a tool to overcome chemical residue problems on fresh vege-
tables. Other uses in agriculture and on forests and ornamentals
was limited.
Inconsistency in field efficacy, however, remained a problem with
early formulations. Investigators began examining environmental
factors that may have been responsible. Cantwell and Franklinz8
reported that B . thuringiensis spores were very susceptible to ul-
traviolet radiation. However, review of the literature indicates that
although observed in the laboratory, observations through harvest
in the field failed to indicate an appreciable deleterious effect on
efficacy.29Nevertheless, recommendations to include an ultraviolet
 PRODUCTION AND UTILIZATION OF B. THURZNGZENSZS 1303

absorber in B . thuringiensis formulations have consistently been

made. Unfortunately, little else was done to improve the physical
formulations being sold.
The lack of effort to improve formulations stemmed directly from
the failure of B . thuringiensis to achieve wide grower acceptance.
This deficiency was due to lack of product consistency against
susceptible species, the incompatibility of existing formulations with
conventional application systems, and problems in grower educa-
tion.
In 1969, the discovery of the HD-1 strain of B . thuringiensis
provided a needed impetus to new formulation research. This strain
was ca. 16x more potent than previous strains being used in com-
mercial production. This was substantiated by Abbott Laboratories,
IMC, Inc., and Nutrilite, and all companies eventually adopted the
strain for their respective products. Concurrently, the Environmen-
tal Protection Agency (EPA) adopted a new profile in registration
of microbials and Dr. Howard Dulmage, in cooperation with indus-
try, succeeded in getting the International Unit adopted as the
standard of potency for B . thuringiensis instead of the spore count.
This toxicity unit provided the basis upon which quality control
could be built. By standardizing against a national standard, HD-I-
S-1971, lot-to-lot variation in the products was eliminated.
Because of this the products began to provide good, consistent,
field results. In the past 10 years B . thuringiensis has become the
standard control agent for cabbage looper, Trichoplusia ni, and
other cabbage worms on vegetables. It has become the material
against which new chemical candidates are compared. Grower ac-
ceptance, excellent efficacy, and the advent of improved formula-
tions have resulted in the worldwide use of in excess of 2 x lo6 lb
product/yr.
Uses and Applications
Bacillus thuringiensis is used against a wide range of lepidopter-
ous defoliators in cotton, soybeans, forests, grapes, tobacco, fruits,
nuts, and vegetables. The organism is registered to control 92 spe-
cies of insects worldwide. In many cases, it is the cornerstone upon
which an integrated pest management program is based. The uses
of B . thuringiensis are still-being extended as growers are educated
to the unique properties of this bacterium. Also with the advent of
new strains, particularly those which destroy insects affecting man
and animals, entirely new areas of growth and penetration are ex-
pected.
1304 COUCH AND ROSS

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Accepted for Publication October 3, 1979