B

BACILLUS

Contents
Introduction
Bacillus anthracis
Bacillus cereus
Geobacillus stearothermophilus (Formerly Bacillus stearothermophilus)
Detection by Classical Cultural Techniques
Detection of Toxins

Introduction
I Jenson, Meat & Livestock Australia, North Sydney, NSW, Australia
Ó 2014 Elsevier Ltd. All rights reserved.
This article is a revision of the previous edition article by Michael K. Dahl, volume 1, pp 113–119, Ó 1999, Elsevier Ltd.

Introduction man as pathogens, causes of food spoilage, or beneficial in

The genus known as Bacillus to traditional food microbiology
has disaggregated in the past 20 years, as molecular taxonomy
has exploded the genus to create new genera and families.
Despite these changes in taxonomy, the significance of the
bacteria found previously entirely in the genus Bacillus has
changed little for food microbiologists. The collective noun
‘bacilli’ will be used to denote this phylogenetically diverse
collection of microbes. A number of species of bacilli are
significant foodborne pathogens and spoilage organisms.
Certain strains of bacilli also may be used as insecticides, as
sources of enzymes for food processing, and as probiotics.
The characteristics that result in these organisms being
significant in food microbiology include their ability to grow
over a wide range of temperatures and pH, lack of complex
nutritional requirements, ability to survive food-processing
conditions such as the application of high temperature,
production of extracellular enzymes that result in the degra-
dation of food components, and production of polymers that
change the sensory characteristics of food.
This article describes the current nomenclature of the bacilli
of interest to food microbiology and elucidates the main
characteristics of these organisms that make them significant to
food production and processing. Finally, the significance of
these organisms as pathogens, probiotics, and sources of food-
grade enzymes will be discussed.
Taxonomy
The application of molecular taxonomy has resulted in the
genus becoming rather less heterogeneous as species have
been moved to new genera and even new families. Over the
same time, many new species have been described. Current
classifications are based firmly on a phylogenetic approach
using 16S rRNA gene sequences as the basis for defining
families and genera. Species of interest to food microbiologists
are now to be found in several genera and in more than one
family, but only a few new species are of interest to food
microbiologists.
Within the phylum Firmicutes and order Bacillales, families
of interest to food microbiologists (and relevant to this article)
include Bacillaceae, Paenibacillaceae, and Alicyclobacillaceae.
The Firmicutes include Gram-positive bacteria with a low DNA
mol% GþC and have rigid cells walls containing muramic acid.
Bacillales is distinguished from other orders based on its rRNA

Encyclopedia of Food Microbiology, Volume 1 http://dx.doi.org/10.1016/B978-0-12-384730-0.00018-5 111

112 BACILLUS j Introduction
sequence homology. All of the families of interest to food
microbiologists contain species formerly classified as Bacillus
and in this article are referred to as bacilli. No phenotypic
characteristics will clearly allow the differentiation of one
family from another, although a combination of characteristics
may be suggestive of a particular family.
The family Bacillaceae now has 19 genera. The genus Bacillus
incorporates many species of Gram-positive, rod-shaped
bacteria, which are able to grow under aerobic and anaerobic
conditions (i.e., they are facultative) and thus differ from
Clostridium spp., which are strictly anaerobic. This differentia-
tion is of practical importance, but it should be noted that
Clostridium is not a member of the order Bacillales. The family
Bacillaceae will undergo further taxonomic rearrangement
based on significant differences in 16S rRNA gene sequences of
some genera. Bacillus and Geobacillus are part of the Bacillaceae
sensu stricto. Geobacillus generally have a more limited range of
growth; for example, they don’t grow at lower temperatures
(i.e., they are thermophiles), higher salt concentrations, or
extremes of pH.
The genus Bacillus consists of a group of Gram-positive
(although may sometimes stain Gram negative) endospore-
forming rods that grow aerobically and usually produce cata-
lase. At present, the genus Bacillus encompasses more than 140
species. These species are widespread in nature and can be
isolated from food, soil, water, animals, and plants.
The members of the family Paenibacillaceae that may be of
interest to food microbiologists include species causing defects
in canned foods and diseases of honeybees. They are meso-
philes with spores that swell the sporangium.
The family Alicyclobacillaceae, with a single genus, Alicy-
clobacillus, contains many species that are found in the envi-
ronment, which often are considered to be extremophiles.
Some species find their way into food and cause taints.
The species of relevance to food microbiology, and relevant
characteristics are summarized in Table 1.
Genetics
Bacillus subtilis is probably the best understood of Gram-positive
prokaryotes. In the late 1950s, John Spizizen successfully
demonstrated the genetic transformation of a particular B. sub-
tilis isolate using purified DNA. Several members of the Bacillus
genus, the best studied of which is B. subtilis, demonstrate
natural competence for DNA uptake under certain conditions.
Before sporulation initiation, about 10–20% of cells in a culture
express competence in the postexponential growth phase under
defined growth conditions. Such competent cells efficiently
bind, process, and internalize available exogenous high-
molecular-weight DNA. The DNA can originate either from
chromosomal DNA or DNA fragments, which must integrate
themselves into the host chromosome to be replicated, or from
plasmid DNA, which can endure and replicate as extrachro-
mosomal DNA in the cytoplasm if it contains a functional origin
of replication. Studies of transformation provided a foundation
for a series of intensive studies of metabolism, gene regulation,
bacterial differentiation, chemotaxis, and starvation.
The complete sequence of the genome of B. subtilis was
determined in 1997, which has facilitated further investigations.
The genome of B. subtilis is characterized by significant dupli-
cation of many families of genes, with many genes that code for
enzymes allowing for the utilization of a wide variety of carbon
sources, particularly those found in plants, and multiple secre-
tion systems. Many genes also are associated with synthesis of
secondary metabolites, such as antibiotics. Many prophage and
parts of prophage genes also are present, indicating the impor-
tance of horizontal gene transfer in this species.
The genomes of many Bacillus and Bacillus-like species have
now been sequenced and information can be found at http://
genodb.pasteur.fr/cgi-bin/WebObjects/GenoList
Growth and Survival
The feature that distinguishes bacilli taxonomically is the
formation of dormant structures, formed from within the
bacterial cell, called endospores. These dormant structures also
are significant in food microbiology because they are resistant
to heat and to desiccation. Under suitable conditions, endo-
spores will germinate and the resultant vegetative cells will
grow and reproduce by binary fission. The life cycle and
survival of bacilli are dependent on their Gram-positive cell
wall structure and ability to form endospores.
The cell wall of Bacillus consists of peptidoglycan and is
composed of up to about 30 layers. The peptidoglycan is a het-
eropolymer of glycan cross-linked by short peptides. Peptide
chains are always composed of alternating L- and D-amino acids.
The Gram-positive bacteria, including Bacillus, reveal a highly
varied peptidoglycan composition and structure. About a 100
different types have been described. Therefore, cell wall
composition often has been a useful criterion in taxonomy.
Spore formation in Bacillus takes place when the cell culture
reaches the stationary growth phase. Sporulation may be
induced by nutritional deprivation, or cell density and is
affected by numerous factors, such as temperature, pH, aera-
tion, and availability of various nutrients. During the sporu-
lation process, a vegetative cell (the progenitor) gives rise to
two specialized cells that differ in cell type both from each
other and from the parent cell. In some cases, this process is
associated with the synthesis of useful products, such as insect
toxins and peptide synthetases creating peptide antibiotics.
The sporulation process is initiated at the end of the expo-
nential growth phase. The development of the endospore
formation involves an energy-intensive pathway and requires
the production of a complex morphological structure. External
(and presumably also internal, however, partially unknown)
signals force the cell to respond by inhibiting cell division and
initiating the sporulation process. In contrast to vegetative
growth, sporulation gives rise to an asymmetrically positioned
septum, which partitions the developing cell into compartments
of unequal sizes. The smaller part is the forespore, which in its
subsequent development exhibits a biochemical composition
and structure completely different from the remaining mother
cell. During the sporulation process, several genes are activated
sequentially; this selected gene activation is induced by the
communication of mother cell and forespore, by signals trans-
ferred across the septum. In turn, the forespore is engulfed by the
other cell, resulting in the endospore, initially within the mother
cell, but subsequently the mother cell dies by cell lysis.
Table 1 Characteristics of bacilli relevant to food microbiology Bacillus species are arranged so that similar strains are generally grouped together

Growth
Temperature
d Significance of the
Spores oval Spores Anaerobic þ NaCl Maximum Casein Starch Gelatin species in food
Family Species or cylindrical spherical growth d Growth pH Growth % degradation degradation degradation microbiology

Bacillaceae Bacillus subtilis þCentral, not swelling – 5–10 6–8 7, Some strains 10 þ þ þ Rope in bakery products, human
20–40 illness, production of some
50–55 fermented soy products such
as natto and kinema; animal
and human probiotic
Bacillus þCentral, not swelling – 5–10 7 Some 5, Some strains 10 þ þ þ Production of a-amylase,
amyloliquefaciens 20–50 strains 10 metalloprotease
55–60
Bacillus licheniformis þCentral to terminal, þ – 6–7 7, Some strains 10 þ þ þ Rope in bakery products, human
not swelling 20–50 illness, flat sour defect in
55 canned foods; animal
probiotic. Production of
a-amylase, serine protease
Bacillus circulans þCentral or terminal, þ nd Some strains 5 nd þ þ d Production of xylanase
swollen 30–50 6–9
55 Some strains 10
Bacillus pumilus þCentral, not swelling – 5–10 Some strains 5 10 þ – þ Potential foodborne pathogen
20–40 6–9
50
Bacillus cereus þCentral, not swelling þ 10 6–7 5 þ þ þ Human illness; bitty cream;
20–40 Some strains 7 animal and human probiotic,
– phospholipase
Bacillus mycoides þCentral, not swelling þ 10 6–7 7 þ þ þ Member of the B. cereus group
20–30
40
Bacillus pseudomycoides þCentral, not swelling þ – 6–7 7 þ þ þ Member of the B. cereus group
10–40
–
Bacillus thuringiensis þCentral, not swelling þ 10 7 7 þ þ þ Insect pathogen, potentially
20–40 foodborne pathogen
–

BACILLUS j Introduction
Bacillus anthracis þCentral, not swelling þ – 6–7 7 þ þ þ Anthrax in herbivores and man
20–40
–
Bacillus þCentral, not swelling þ 5 6–7 5 þ þ þ Food spoilage
weihenstephanensis 10–30 Some strains 7
40
Bacillus coagulans þCentral/terminal, þ nd 5–10 2 – þ d Aciduric flat sour defect of
swollen 30–40 canned foods, tomato juice
50–55 and milk, human and animal
probiotic. Production of
glucose isomerase

(Continued)(Continued)

113
 114
Table 1 Characteristics of bacilli relevant to food microbiology Bacillus species are arranged so that similar strains are generally grouped togetherdcont'd

Growth
Temperature

BACILLUS j Introduction
d Significance of the
Spores oval Spores Anaerobic þ NaCl Maximum Casein Starch Gelatin species in food
Family Species or cylindrical spherical growth d Growth pH Growth % degradation degradation degradation microbiology

Bacillus alcalophilus þCentral – – 8–9 2 þ þ þ Production of serine protease

10–40 Some strains 10
–
Bacillus clausii þCentral variable nd – 7–8 7, Some 10% þ þ þ Potential probiotic
swelling 20–50
–
Bacillus smithii þCentral/ þ – 6–7 – w – Aciduric flat sour defect of
terminal, variable 30–55 canned foods, evaporated
swelling 65 milk, cheese, sugar beet juice
Geobacillus þSubterminal to – 35 6–8 4%, Some strains 5% d/w þ þ Thermophilic flat sour defect of
stearothermophilus terminal not 40–70 canned foods. Production of
usually swollen 75 a-amylase, glucose kinase,
glucose-6-phosphate
dehydrogenase,
phosphotransacetylase
Lysinibacillus sphaericus þTerminal – 10 Some strains 6 5 d – d Insect pathogen
swollen 20–30 7–9
40
Paenibacillaceae Paenibacillus polymyxa þTerminal, swollen þ 30 opt 7 Some strains 2% þ þ þ Flat sour defect of canned foods
Paenibacillus macerans þTerminal, swollen þ 30 opt, some 7 nd – þ þ Flat sour defect of canned foods
strains 50
Paenibacillus larvae þCentral or terminal, þ 28–37 opt nd 2% þ – þ Insect pathogen – American
swollen foulbrood of honeybees
Paenibacillus popilliae þCentral, swollen þ 28–30 opt nd 3% – – – Pathogenic to the Japanese
beetle
Brevibacillus brevis þSubterminal swollen – 20 6–8 Some strains 2% þ – þ Potential foodborne pathogen
30–40
50
Alicyclobacillaceae Alicyclobacillus þTerminal – 45–70 2–6 2% Tainted acidic foods
acidocaldarius 60–65 opt (3–4 opt)
Alicyclobacillus þTerminal/subterminal – 35–55 2.2–5.8 4% Tainted acidic foods
acidoterrestris 42–53 opt (4 opt)

þ, 85% positive; d, depends on strain 16–84% positive; –, Less than 15% positive; v, variation with strains; w, weak; opt, optimum; nd, no data.
Sources: Cutting, S.M., 2011. Bacillus probiotics. Food Microbiology 28, 214–220. de Vos, P., Garrity, G.M., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A., Schleifer, K.-H., Witman, W.B. (Eds.), Bergeys Manual of Systematic Bacteriology,
vol. 3. The Firmicutes, second ed. Springer, New York. Glick, B.R., Pasternak, J.J., Patten, C.L., 2010. Molecular Biotechnology: Principles and Applications of Recombinant DNA, fourth ed. Washington: ASM Press. Jenson, I., Jensen, N., Hyde,
M., 2001. Gram positive aerobic sporeforming rods. In: Moir, C.J., Andrew-Kabilafkas, C., Arnold, G., Cox, B.M., Hocking, A.D., Jenson, I. (Eds.), Spoilage of Processed Foods: Causes and Diagnosis. Australian Institute of Food Science and
Technology (NSW Branch) Food Microbiology Group, Sydney, pp. 271–294.

The spore consists of layers of modified peptidoglycan and
proteins that are unique to spores. The spore is relatively dry
and contains large amounts of dipicolinic acid as well as
divalent cations. The structure and composition of spores and
their metabolic inactivity is responsible for the long dormancy
and resistance to heat and desiccation.
The process of conversion of the endospore to vegetative cell
involves three steps: activation, germination, and outgrowth. In
some species, mild heat treatment is able to activate the process
of germination. Even without activation, germination can
occur when environmental conditions are suitable for cell
growth. During germination, the spore cortex is degraded, the
spore becomes hydrated, and dipicolinic acid and minerals are
excreted. Hydration allows conformation of DNA, ribosomes,
and enzymes to be restored, which then allows metabolic
activities to resume. Depending on the type of spore formation
observed, we distinguish among the following:
l species producing oval endospores that distend the mother
cell
l species producing oval endospores that do not distend the
mother cell
l species producing spherical endospores
The morphological characteristics of the spores of various
bacilli are summarized in Table 1.
Spores are more resistant than vegetative cells to heat,
freezing, high pressure, desiccation, g radiation, ultraviolet
light, chemicals, and extreme pH.
The temperature resistance of spores is a significant feature
of bacilli and their significance in food processing. This resis-
tance is related to the maximum growth temperature of vege-
tative cells. The various species of bacilli grow at temperatures
that encompass a wide range from psychrotolerant species to
thermophiles. Strains or species with a higher maximum
growth temperature are more temperature tolerant, and thus
they require exposure to a higher temperature to obtain the
same decimal reduction time (inactivation rate). Temperature
resistance is modulated by the actual growth temperature of the
cells before sporulation, where again, growth at higher
temperatures leads to greater heat resistance. Sporulation at
neutral pH also favors heat resistance of the spores. Clearly
some of these factors can lead to deviations from the heat-
resistance data (D- and z-values) published in the literature and
can be significant to various food processes.
Metabolism
The behavior of bacilli in food, and the resulting spoilage or
growth to levels that can cause human illness, is a result of their
nutritional requirements, degradation and metabolism of
nutrients, and the production of polymers.
Most bacilli of significance in foods and food environments
have nonexacting nutritional requirements. Paenibacillus spp.
may be more fastidious, which means that these species are
more likely to contaminate food via nutrient-rich rather than
nutrient-poor soils, but once in a nutritionally rich food envi-
ronment, they are unlikely to behave differently than other
bacilli. Some strains of Bacillus thuringiensis may require less
nutritious media. Alicyclobacillus sp. do not have exacting
BACILLUS j Introduction 115
nutritional requirements, but they do require media with low
pH (Table 1).
Many bacilli produce extracellular hydrolytic enzymes
essential for the breakdown of polysaccharides or oligosac-
charides, nucleic acids, proteins, and lipids (Table 1). The
resulting products can be used as carbon sources, nitrogen
sources, energy sources, and electron donors. They also contain
hydrolytic enzymes in the cytoplasm, however, which prepare
carbon sources to enter glycolysis by further hydrolytic, phos-
phorylation, and isomerization reactions.
Glucose is readily utilized as a sole carbon source that
usually is fermented. Many mesophilic bacilli can grow
anaerobically and ferment glucose to produce 2,3-butanediol,
glycerol, and carbon dioxide. Small amounts of lactate,
ethanol, diacetyl, or acetate may be produced by some species.
Bacilli may utilize a range of nitrogen sources, including
ammonium ions, urea, and amino acids. Fixation of atmo-
spheric nitrogen may occur in Bacillus cereus, Bacillus lichen-
iformis, and Paenibacillus species.
Levan and dextran are sucrose polysaccharides produced by
some species. When a product is spoiled by Alicyclobacillus, the
juice products develop a disinfectant-like odor or flavor due to
the production of guaiacol, probably from the degradation of
ferulic acid.
Significance in Food Spoilage
The bacilli may cause significant spoilage problems. Bacilli are
found in soil and consequently also in water and air. Soil is
considered to be the primary habitat for most bacilli.
Contamination of food generally is considered to be via the
entry of soil, air, or water.
Bacilli are particularly noted as spoilage organisms in heat-
treated (including, retorted) foods. Bacilli may not dominate
the microbial population of a food before heat treatment but
treatment failure and the absence of competitive microorgan-
isms can result in spoilage of shelf-stable foods by bacilli.
Bacilli may be responsible for the spoilage of dairy and bakery
products, among others, that have undergone mild (pasteuri-
zation) heat-treatment processes, and the products are held at
ambient or refrigeration temperatures for long periods of time.
These organisms survive in food processing because of
thermal tolerance resulting from their thermophilic nature or
spore formation, but this does not explain why they spoil foods.
Spoilage occurs primarily due to the ability of many bacilli to
grow under a wide range of conditions and due to the fact that
they possess metabolic capabilities to access a wide variety of
substrates and produce a range of undesirable end products.
Many bacilli growth over a wide range of conditions. Most
bacilli grow at temperatures under 20  C and above 40  C, with
some growing well at temperatures above 50  C. Strains of
some species appear to be able to adapt to grow at temperatures
outside the usual range (Table 1). Although some bacilli grow
in a narrow range of pH around neutral, some are acidophilic
(Alicyclobacillus) and others grow over a wide range of pH
(Bacillus coagulans). Some species of bacilli are able to grow at
high salt concentrations (Table 1).
The ability of many species to grow under anaerobic condi-
tions also provides opportunities to spoil foods. Although
116 BACILLUS j Introduction
B. subtilis usually is regarded as an aerobe, it actually is able
to grow anaerobically using nitrate or nitrite as an electron
acceptor.
The widespread ability to degrade polymers – such as starch,
fats, gelatin, or casein–provides bacilli with the opportunity to
cause changes in the structures of a number of foods as well as
leads to end products of metabolism that are unpleasant to
consumers (Table 1). For example, the production of amylo-
lytic enzymes by B. subtilis leads to the degradation of starch,
which results in stickiness in bread and stringy strands when
the bread is pulled apart (rope). The fermentable carbohydrate
released (glucose) is fermented to mixed acids and alcohols,
which result in estery odors. Another example is the phos-
pholipase of B. cereus, which cleaves fatty acids from lipids
resulting in bitter flavors and unstable fat globules when milk is
heated (e.g., in a beverage), so-called bitty cream. Spoilage of
vegetables may be due to such bacilli as B. subtilis and Paeni-
bacillus sp. in potato rot.
Bacilli also may form polymers of glucose or sucrose. These
polysaccharides (dextrans and levans) contribute to sliminess
in spoiled foods and stringiness (rope formation) in spoiled
bread and bakery products. Levan production by B. subtilis can
cause processing problems in sugar refining and B. licheniformis
may cause ropiness in alcoholic beverages, such as cider.
Foodborne Disease
Several species of bacilli, almost all in the genus Bacillus, are
implicated as human foodborne pathogens. Bacillus cereus and
Bacillus anthracis, are well recognized as foodborne pathogens,
whereas the evidence for the pathogenicity of B. subtilis and
B. licheniformis is less well developed. Illness has been reported
due to other species, including Bacillus pumilus, B. thuringiensis,
and Brevibacillus brevis.
Bacillus cereus and B. anthracis are closely related organisms,
distinguishable by a few somewhat-variable characteristics, but
ultimately by possession of genes that determine pathogenicity.
Bacillus cereus possesses a chromosomally encoded b-lactamase,
whereas B. anthracis is virtually always penicillin sensitive. Not
all strains of B. cereus are pathogenic for humans. Two toxins,
one causing diarrhea and the other provoking vomiting (emetic
toxin), may be produced. The diarrheal syndrome often is
associated with protein-rich foods, whereas the emetic
syndrome often is associated with starchy foods, custards, and
dairy products. The production of toxin appears to be associated
with certain genetic clusters of strains, with some producing one
toxin and some the other. The closely related species Bacillus
weihenstephanensis does not appear to produce either toxin.
Human pathogenic potential exists in some B. thuringiensis
strains and has been implicated in some human illness. Strains
that are used as commercial insecticides, however, appear to
have low ability to produce enterotoxin. Bacillus anthracis
possesses three toxin genes that are located extrachromosomally
on a large plasmid. Bacillus anthracis causes the disease anthrax,
primarily as disease of animals, which spreads to humans
usually through minor breaks in the skin or mucous membranes
from wool or hairs from infected animals. Cutaneous anthrax
first appears as a papule, which develops into a vesicle and after
2–6 days into a black eschar; 5–20% of untreated cases are fatal.
Gastrointestinal or oropharyngeal anthrax also may occur in
humans due to the ingestion of milk or meat from infected
animals. Antemortem and postmortem veterinary examination
should exclude anthrax-affected animals from the food chain,
but in areas of the world where veterinary examination is
nonexistent, gastrointestinal anthrax may occur, requiring
surgery to remove the affected part of the colon.
Foodborne illness due to other Bacillus species has been
reported infrequently. Bacillus subtilis or B. licheniformis
have been implicated most frequently. Bacillus pumilus,
B. thuringiensis, and Brevibacillus brevis also have been impli-
cated in human illness. Quite possibly, lack of definitive
methods for these species, and the inability to easily distin-
guish these species from other bacilli that may be present in
food has led to these species being underrecognized. A few
researchers have collected a number of cases. A wide range of
foods appears to be implicated, usually involving relatively
mild heating steps, which may create an environment in which
these species can grow easily to high levels. High levels have
been sometimes found in implicated food and sometimes in
the absence of other known agents of foodborne disease. Both
emetic and diarrheal episodes have been reported, which are
usually of short duration, with onset within an hour to several
hours after consumption of the implicated food.
Insect Control
Different variants of B. thuringiensis, Paenibacillus popilliae, Pae-
nibacillus larvae, B. cereus, Lysinibacillus sphaericus, and other
related species are pathogenic to insects. The use of these strains
for microbial insect control offers the advantage of being safer
than the more toxic chemical control agents. Furthermore, they
have relatively slight effects on the ecological balance of the
environment due to their specificity for insect larvae. The
microbial insecticide is composed of, at least in the case of
B. thuringiensis, spores and crystalline proteins that, when
ingested by larvae, cause gut paralysis, probably by upsetting the
ionic balance of the gut. The spore survives its passage through
the gut, penetrates the weakened midgut wall, and multiplies in
the hemolymph. Death results from either intoxication or
septicemia. High selectivity and the absence of harmful side
effects on plants, warm-blooded animals, or humans give many
of the Bacillus products an advantage over other insecticides.
Several insect-specific pathogens are produced commercially for
use as microbial pesticides. Bacillus thuringiensis is produced
commercially as an insect larvicide and has been used world-
wide to control damage to crops, trees, and ornamental plants.
During endospore formation, this bacterium produces toxic
protein crystals (Bt toxin) that make it a good pesticide. Most of
the toxin genes of B. thuringiensis are located on conjugative
plasmids, which are transmissible by conjugation between
B. thuringiensis and B. cereus under laboratory conditions.
Paenibacillus popilliae causes a fatal illness called milky
disease in Japanese beetle larvae. After ingestion by the larvae,
B. popilliae germinates in the gut, begins to multiply, and
invades the hemolymph. After about 10 days, a typical milky
appearance is observed due to the massive numbers of bacteria.
Bacillus cereus strains often are pathogenic for insects. They
produce phospholipase C, an a-exotoxin that permits the

bacteria to pass through the barrier of the intestinal epithelial
cells. Subsequent penetration into the hemolymph followed by
multiplication kill the insect.
Probiotics
Bacillus species are sometimes used as probiotics in both animals
and humans. Bacillus subtilis, Bacillus clausii, B. cereus, B. coagulans,
and B. licheniformis have been the most extensively examined. It
is clear that few scientific studies have been performed on the
potential of these species as probiotics, especially compared with
the application of lactic acid bacteria. Bacillus have the obvious
advantage over other potential probiotics that they can be
produced efficiently and cost effectively by drying and survive
well through shelf life. They also will survive gastric acidity and
survive into the bowel, the reputed site of action. At least some
strains are able to germinate and reproduce in the human
gastrointestinal tract. A number of products are registered for
human use and also for animal use (pigs, poultry, calves,
aquaculture). Most products on the market do not have exten-
sive clinical trial data. Claims often relate to extragastrointestinal
effects, such as alleviation of allergy symptoms and rheumatoid
arthritis symptoms, but claims for gastrointestinal efficacy are
made for some products. The mechanisms by which bacilli may
be effective as probiotics is not known. Some strains have been
shown to produce extracellular proteases.
Natto is a B. subtilis–fermented soybean product, native to
Japan, for which health benefits often have been ascribed. It is
popular as a breakfast food. Natto has a strong odor and flavor
and a viscous, stringy texture. In part, this is due to the
production of a cell capsule of poly-g-glutamic acid in stationary
phase. Some B. cereus probiotic products are produced from
strains that produce enterotoxin or at least contain enterotoxin
genes.
Enzyme Production
The genus Bacillus encompasses species often used for the
production of metabolites and enzymes by fermentation. This
is partly due to the fact that most are excellent protein and
metabolite secretors and are easy to cultivate. The tremendous
advances in molecular biology have increased the use of Bacillus
spp. in heterologous gene expression.
Nonpathogenic Bacillus strains are used both in food pro-
cessing and industrial fermentation. The numerous products
accepted as safe include enzymes for food and drug processing,
as well as foods produced from these strains.
Bacillus species are used to manufacture commercially
important enzymes (Table 1). For example, amylases are used
BACILLUS j Introduction 117
for the production of glucose from corn, wheat, or potato
starch. The resulting glucose can be converted by glucose
isomerase to a glucose–fructose mixture, which has a sweeter
taste than either glucose or sucrose. This enzymatic process
therefore has become important for the industrial production
of sugar from starch, either as a substrate for subsequent
fermentation to ethanol, or as a sweetening agent in soft drinks
and other foods. In principle, these reactions can be catalyzed
separately by enzymes, which operate sequentially in the
conversion reactions. These reactions are composed of three
principal steps:
l Thinning reaction, in which the starch polysaccharides are
attacked by a-amylase, shortening the chain and reducing
viscosity.
l Saccharification, which produces glucose from the shortened
polysaccharides catalyzed by the glucoamylase.
l Isomerization, which converts glucose into fructose, cata-
lyzed by the glucose isomerase.
Cellulases, lipases, and proteases also may be produced
from B. subtilis and B. licheniformis.
See also: Bacillus: Bacillus cereus; Geobacillus
stearothermophilus (Formerly Bacillus stearothermophilus);
Bacterial Endospores.
Further Reading
Cutting, S.M., 2011. Bacillus probiotics. Food Microbiology 28, 214–220.
de Vos, P., Garrity, G., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A., Schleifer, K.-H.,
Witman, W.B. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 3. The
Firmicutes, second ed. Springer, New York.
Guinebretière, M.-H., Thompson, F.L., Sorokin, A., et al., 2008. Ecological diversifi-
cation in the Bacillus cereus group. Environmental Microbiology 10, 851–865.
Jenson, I., Jensen, N., Hyde, M., 2001. Gram positive aerobic sporeforming rods.
In: Moir, C.J., Andrew-Kabilafkas, C., Arnold, G., Cox, B.M., Hocking, A.D.,
Jenson, I. (Eds.), Spoilage of Processed Foods: Causes and Diagnosis. Australian
Institute of Food Science and Technology (NSW Branch) Food Microbiology Group,
Sydney, pp. 271–294.
Jenson, I., Moir, C.J., 2003. Bacillus cereus and other Bacillus species.
In: Hocking, A.D. (Ed.), Foodborne Microorganisms of Public Health Significance,
sixth ed. Australian Institute of Food Science and Technology (NSW Branch) Food
Microbiology Group, Sydney, pp. 445–478.
Kramer, J.M., Gilbert, R.J., 1989. Bacillus cereus and other Bacillus species.
In: Doyle, M.P. (Ed.), Foodborne Bacterial Pathogens. Marcel Dekker, NY,
pp. 21–70.
Kunst, F., Ogasawara, N., Moszer, I., et al., 1997. The complete genome sequence of
the gram-positive bacterium Bacillus subtilis. Nature 390, 249–256.
Palop, A., Mañas, P., Condón, S., 1999. Sporulation temperature and heat resistance
of Bacillus spores. A review. Journal of Food Safety 19, 57–72.
Warth, A.D., 1978. Relationship between the heat resistance of spores and the
optimum and maximum growth temperature of Bacillus species. Journal Bacteri-
ology 134, 699–705.