Citric Acid see Fermentation (Industrial): Production of Some Organic Acids (Citric, Gluconic, Lactic, and Propionic)
Citrobacter see Salmonella: Detection by Immunoassays
CLOSTRIDIUM
Contents
Introduction
Clostridium acetobutylicum
Clostridium botulinum
Clostridium perfringens
Clostridium tyrobutyricum
Detection of Enterotoxin of Clostridium perfringens
Detection of Neurotoxins of Clostridium botulinum
Introduction
HP Blaschek, University of Illinois at Urbana-Champaign, Urbana, IL, USA
Ó 2014 Elsevier Ltd. All rights reserved.
Introduction
Characteristics of the Genus Clostridium
The genus Clostridium contains physiologically and genetically
diverse species involved in the production of toxins as well as
acids and solvents. The broad range of mol% GþC values
together with 16S rRNA cataloging demonstrates a high degree
of phylogenetic heterogeneity within the genus Clostridium.
Cato and Stackebrandt indicated that the genus does not
include a phylogenetically coherent taxon. From an evolu-
tionary standpoint, members of this genus appear to have
evolved during ancient times, perhaps during the anaerobic
phase of evolution.
Species within the genus Clostridium have both medical and
industrial significance. The genus Clostridium originally was
described in 1880. Because of the observed heterogeneity, it
should not be surprising that the genus is quite large, on the
order of 100 species. The clostridia are ubiquitous and are
commonly found in the soil, marine sediments, and animal and
plant products. They typically are found in the intestinal tract of
humans and in the wounds of soft tissue infections of humans
and animals. To be included within this genus, the isolate must
be anaerobic or microaerophilic; be able to produce an endo-
spore-forming rod, Gram-positive, or Gram variable; and be
unable to carry out dissimilatory sulfate reduction. There is
444 Encyclopedia of Food Microbiology, Volume 1
considerable interspecies variability in these observed criteria,
and intraspecies Gram-stain variability appears in some cases to
be related to the age of the culture. Most clostridia are motile
via peritrichous flagella; however, for some species such as
C. perfringens, motility has not been observed.
Clostridial spores appear to be produced only under
anaerobic conditions, and in some species, sporulation occurs
with considerable difficulty. In certain cases, special media have
been formulated to promote sporulation. Depending on the
species, endospores may occur in a central, subterminal, or
terminal positions and, because of the slender nature of the
mother cell sporangium, cells containing spores appear
swollen, unlike the thicker bacilli. The location of the endo-
spores within the cell has important taxonomic value. The heat
resistance of clostridial spores is also a function of the species;
however, because of their foodborne association and patho-
genic nature, the greatest concern is with the spores produced
by C. botulinum and C. perfringens. These spores may be able to
survive routine cooking procedures and germinate and resume
vegetative growth once nutritionally and environmentally
appropriate conditions return. Because of the spore-forming
capability of this foodborne pathogen, C. botulinum is regarded
as the ‘target microorganism’ for the development of appro-
priate time–temperature heat treatment relationships for can-
ned food products.
http://dx.doi.org/10.1016/B978-0-12-384730-0.00067-7

Table 1 Species of Clostridium involved in causing diseases
Species Diseases
C. perfringens Food poisoning, gas gangrene, necrotic
enteritis, minor wound infection
C. tetani Tetanus
C. botulinum Botulism food poisoning
C. difficile Pseudomembranous colitis enterocolitis
C. novyi Gas gangrene
C. histolyticum Gas gangrene
C. septicum Gas gangrene
Species within the genus Clostridium produce a wide diver-
sity of exoproteins, many of which function as virulence
factors. Some of these proteins are antigenic in nature and
some have associated enzyme activity. An overview of the
major and minor antigens produced by C. perfringens is given in
the article on C. perfringens. Table 1 lists a representative group
of clostridial species that cause various diseases. The most
important species with respect to human disease include
C. botulinum, C. perfringens, C. tetani, and C. difficile. The role of
toxins produced by these species in causing disease has been
well characterized.
From the standpoint of metabolism, there appears to be a
delineation between clostridia that are principally saccharolytic
and those described as proteolytic. Genetic studies have
demonstrated that strains falling into these two groups are
unrelated with respect to DNA similarity. Following growth on
carbohydrates, the clostridia usually produce mixtures of
alcohols and organic acids. The clostridia use the Embden–
Meyerhof–Parnas pathway for breakdown of monosacc-
harides. Although carbohydrates appear to be the preferred
carbon source, metabolism of alcohols, amino acids, and other
organic compounds may also occur. Purines and pyrimidines
have also been shown to be fermented by various species of
clostridia. The industrial utility of the clostridia is enhanced by
their ability to degrade and utilize a diverse group of poly-
saccharides. Various species of clostridia are able to degrade
polymers (such as cellulose, starch, and pectin) and produce
useful products such as acids and solvents. The ability of
the clostridia to coferment both five and six carbon sugars
bodes well for utilization of biomass as a fermentation feed-
stock. The acetone–butanol–ethanol (ABE) fermentation using
C. acetobutylicum or C. beijerinckii growing on starch or molasses
dominates the history of clostridial fermentations.
For most clostridial species, growth occurs most rapidly
between pH 6.5 and 7.0 and at a temperature of 30–37  C,
although some species, such as C. perfringens have very rapid
growth (generation times as low as 10 min) at temperatures of
40–45  C. There are also a number of thermophilic clostridia
that are able to grow up to a maximum temperature of 80  C.
Because of the industrial potential of hydrolytic enzymes –
such as amylase, pullalanase, and glucoamylase – recovered
from the thermophilic clostridia, these microbes recently
have been the subject of intensive investigation. A list of
representative thermophilic clostridial species is presented in
Table 2.
With respect to the development of genetic systems (gene
transfer, shuttle vectors, etc.) for the clostridia, the model
CLOSTRIDIUM j Introduction 445
Table 2 Optimal growth temperatures of thermophilic Clostridium
species
Species Optimal growth temperature (  C)
C. thermoaceticum 58
C. thermosulfurogenes 60
C. thermocellum 60
C. fervidus 68
C. thermosuccinogenes 58–72
C. thermobutyricum 57
C. stercorarium 65
species have been the foodborne pathogen, C. perfringens
and the solventogenic clostridia, which include principally
C. acetobutylicum and C. beijerinckii. Most of the early plasmid
work initiated 20 years ago was carried out with C. perfringens,
whereas the industrial significance of strains that are able to
produce acetone and butanol has resulted in a renewed
emphasis on genetic systems development in C. acetobutylicum
and C. beijerinckii. The molecular tools developed over the past
20 years now are being used to investigate the mechanism of
toxin production in the pathogenic clostridia (e.g., C. perfringens
and C. botulinum) and to understand the molecular basis
for acid and solvent productions (e.g., C. acetobutylicum and
C. beijerinckii). Recently, a genome-scale metabolic model
(iCM925) of butanol-producing C. beijerinckii was described.
The model can accurately reproduce physiological behavior and
provide insight into the underlying mechanisms of microbial
butanol production. RNA-seq technology has been used to carry
out single-nucleotide resolution analysis of the transcriptome of
C. beijerinckii. The application of these technologies is expected
to allow for the directed metabolic engineering of these indus-
trially significant species.
Selected Clostridial Species
Clostridium perfringens
Clostridium perfringens has been described as the most ubiqui-
tous pathogenic bacterium in our environment. This anaerobic
Gram-positive bacterium is an inhabitant of the soil and the
intestinal tract of both humans and animals. It produces as
many as 12 biologically active toxins. Although primarily
associated with foodborne disease, it is also responsible for
causing gas gangrene, lamb dysentery, necrotic enteritis, and
minor wound infection.
Clostridium botulinum
Botulism food poisoning is caused by the consumption of food
containing heat-labile neurotoxin produced by C. botulinum.
C. botulinum first was isolated in 1895 by E. van Ermangen from
salted ham. The causative microorganism was named Bacillus
botulinus (from the Latin ‘botulus’ meaning sausage). It is
described as an intradietic intoxication in which the exotoxin is
produced by the microorganism during growth on the food.
The types of C. botulinum are identified by neutralization of
their toxins by the antitoxin. There are seven recognized anti-
genic types of C. botulinum, A–G (Table 3). In addition to toxin
446 CLOSTRIDIUM j Introduction
Table 3 Occurrence of C. botulinum types
Location Type Proteolytic
Western United States, Canada A þ
Europe, Eastern United States B þ/
Widespread Ca – Paralysis of birds
Widespread Cb – Forage poisoning Cattle
South Africa, Russia, United States D –
Northern hemisphere E –
Japan F þ
Argentina G þ
production, the types are differentiated on the basis of their
ability to produce proteolytic enzymes. The production of
proteolytic enzymes by C. botulinum when present on food
results in a putrid, unpleasant odor that can be a useful
deterrent to consumption. Although strains of C. botulinum are
variably proteolytic, they are always saccharolytic and are able
to ferment glucose with the production of energy as well as acid
and gas. Most outbreaks (w72%) of botulism have been traced
to home canned foods and vegetables, in particular. These
outbreaks have been traced to foods that have been handled
improperly or insufficiently heated to destroy spores. In the
United States, C. botulinum types A and B are most common,
whereas in Europe, meat products frequently have served as the
vehicle, and botulinal food poisoning primarily is due to type B
strains. C. botulinum is a strictly anaerobic, Gram-positive rod
that produces heat-stable spores that are located subterminally
on the mother cell sporangium. The microorganism is motile
via peritrichous flagella. The neurotoxins produced by
C. botulinum and C. tetani are composed of the most potent
group of bacterial toxins known. The toxins act by inhibiting
the release of neurotransmitters from presynaptic nerve termi-
nals inducing a flaccid paralysis (C. botulinum) or a spastic
paralysis (C. tetani). Although the symptoms induced by the
toxins appear dramatically different, the toxins have similarities
in their structures and modes of action.
Detection of C. botulinum Neurotoxins
The botulinum neurotoxins are simple proteins composed of
only amino acids. These toxins are among the most toxic
substances known. Ingestion of as little as 1–2 mg toxin may
prove fatal. Although the toxins produced by C. botulinum have
all been purified and characterized, type A neurotoxin is best
characterized and was the first to be purified. The complete
covalent structure of the proteolytically processed, fully active
type A neurotoxin has been determined. In addition to being
a neurotoxin, hemagglutinin activity is normally associated
with type A toxin. Hemagglutinin is believed to stabilize the
toxin in the gut. The toxin molecule that is produced by
a toxigenic culture is referred to as a progenitor toxin and
consists of a toxic and an atoxic component. The progenitor
toxin is the precursor of the more toxic derivative toxin. The
progenitor toxins can be converted into the derivative form by
the action of proteases in the digestive tract of the host or via
the direct action of proteolytic enzymes associated with the
microorganism. Unlike staphylococcal toxins, botulinal toxins
Disease Antitoxin
Human Specific
Human Specific
X-react Ca and Cb
X-react with D
Cattle X-react with Cb
Human Specific
Human Specific
No disease Unknown
are heat-sensitive proteins. They are destroyed by boiling for
10 min. Therefore, a food can be rendered nontoxic by heating,
although the cooking of a suspect food is not considered
a worthwhile risk. On the other hand, consumption of low
levels of spores by a healthy adult apparently will do no harm.
Tryptophan has been shown to be required for toxin produc-
tion together with carbon dioxide.
Typically, one portion of the food to be examined is set
aside and examined for the presence of the microorganism, and
the other portion is used in toxicity testing. Food samples
containing suspended solids are centrifuged and the superna-
tant fluid examined for toxin. Solid food is extracted with an
equal volume of gel-phosphate buffer. The macerated food
sample is centrifuged under refrigeration and the supernatant is
used for assay of the toxin. Food samples containing toxins of
nonproteolytic C. botulinum may require trypsin activation to
be detected. In this case, the trypsin-treated preparation is
incubated for 1 h with gentle agitation.
The mouse lethality assay was the first method employed for
detection of toxins produced by foodborne pathogens, and
although still used for assay of botulinal toxins, its use has
become more limited with the advent of alternative assays. The
approach when using the mouse lethality assay is quite
straightforward. Pairs of mice are injected intraperitoneally with
trypsin-treated and untreated preparations. A portion of
untreated supernatant fluid or culture is heated for 10 min at
100  C. All injected mice are observed for 3 days for symptoms
of botulism or death. If, after 3 days, all mice except those
receiving the heated preparation have died, the toxicity test
should be repeated using higher dilutions of supernatant fluids
or cultures. This approach allows determination of the
minimum lethal dose (MLD) as an estimate of the amount of
toxin present. From these data, the MLD per milliliter can be
calculated. The precision of the mouse lethality assay for esti-
mating the activity of C. botulinum toxin has been shown to be of
the order of 5%. Protocols for typing of the toxin involves
rehydrating antitoxins with sterile physiological saline. Antisera
can be obtained from the Centers for Disease Control and
Prevention, Atlanta, Georgia, or from the Food and Drug
Administration, Washington, DC. Various types of monovalent
antitoxins are employed. Mice are injected with the respective
monovalent antitoxins 30–60 min before challenge with
toxic samples. A pair of unprotected mice (no injection of
antitoxin) is injected with the toxic sample as a control. Mice are
observed for 48 h for symptoms of botulism and to record
deaths.

Additional approaches for the detection of botulinal toxins
include gel diffusion, specifically electroimmunodiffusion,
which has a reported sensitivity of five mice LD50 per 0.1 ml
and the polymerase chain reaction (PCR), which has been
applied to detect C. botulinum types A–E toxin genes with
a reported sensitivity of 10 femtogram. Another approach is the
evanescent wave immunosensor to detect type B C. botulinum
toxin. The sensor detects fluorescently tagged, toxin-bound
antibodies. The enzyme-linked immunosorbent assay (ELISA)
system has been used successfully to detect C. botulinum toxins.
For type A C. botulinum toxin, a double-sandwich ELISA
detected 50–100 mice LD50 of type A and less than 100 mice
LD50 of type E. A double-sandwich ELISA using alkaline
phosphatase was able to detect one mouse LD50 of type
G toxin. Clostridium botulinum toxin type A was detected at
a level of nine mice LD50 per milliliter when using a mono-
clonal antibody.
The Solventogenic Clostridia: C. acetobutylicum
and C. beijerinckii
The fermentation of carbohydrates to ABE by the solventogenic
clostridia is well known. For an overview of developments in
the genetic manipulation of the solventogenic clostridia for
biotechnology applications, the reader is referred to the further
reading list. Currently, this value-added fermentation process is
attractive for several economic and environmental reasons.
Prominent among the economic factors is the current surplus
of agricultural wastes or by-products that can be utilized as
inexpensive fermentation substrates. Examples include myco-
toxin-contaminated corn that is unsuitable for use as animal
feed and 10% solids light corn steep liquor, which is a low-
value by-product of the corn wet milling industry.
It has been suggested that the instability of certain sol-
ventogenic genes (ctfAB, aad, adc) may be the cause of strain
degeneration in C. acetobutylicum. Specifically, the genes for
butanol and acetone formations in C. acetobutylicum ATCC
824 were found to reside on a large 210 kb (pSOL1) plasmid
whose loss leads to degeneration of this strain. Eight genes
concerned with solventogenic fermentation in C. beijerinckii
8052 were found at three different locations on the genome.
In C. beijerinckii 8052, genomic mapping studies suggest that
the ctfA gene is localized on the chromosome and is colocated
next to the acetoacetate decarboxylase gene. An examination
of the effects of added acetate on culture stability and solvent
production by C. beijerinckii showed that one of the effects
may be to stabilize the solventogenic genes and thereby
prevent strain degeneration. To examine this hypothesis,
further genetic analysis of the solventogenic genes will need to
be carried out.
Given the dramatic advances and cost reductions in
sequencing technologies over the past decade, sequencing
technology is proposed as a means to identify and characterize
subtle, genomic-level changes that occur in the hyperbutanol-
producing C. beijerinckii BA101 mutant, which was produced
using chemical mutagenesis. Differences observed for the
C. beijerinckii BA101 strain (U.S. Patent 6358717) at the
sequence level can be compared directly to the parent strain.
Determination of the genomic alterations responsible for the
physiology associated with the C. beijerinckii BA101
CLOSTRIDIUM j Introduction 447
hyperbutanol phenotype ultimately will lead to the develop-
ment of a strategy for engineering a strain of C. beijerinckii with
enhanced solvent-producing characteristics for industrial
applications.
The genome of C. beijerinckii is approximately 50% larger
than that of its cousin, C. acetobutylicum. C. beijerinckii
demonstrates a multiplicity of genes for which C. acetobutylicum
many only have one or two copies. This may at least partially
explain the differences between the two species. The size of the
C. acetobutylicum genome was found to be 4.11 Mb, with an
overall GþC ratio of 29.2%. There is an expectation for 4200
genes, and analysis of the sequence has revealed similarity,
although not necessarily functionality, to a number of antibi-
otic-resistant genes, clostridial-toxin genes, and various
substrate hydrolytic genes. It is expected that analysis of the
chromosome sequence will provide important information
regarding the phylogenetic relatedness of the solvent-
producing clostridia.
Recommended Methods of Detection
and Enumeration in Foods
The clostridia generally can be isolated on nutritionally
complex media that are appropriate for the cultivation of
anaerobes. This may, for example, include blood agar
and cooked meat medium. Tryptone–glucose–yeast extract
medium is easy to prepare and can meet the nutritional
requirements of many different species of clostridia. The media
should be reduced, normally by the addition of L-cysteine or
sodium thioglycollate. To selectively recover clostridia from the
soil or intestinal contents, it is useful to heat the sample at
80  C for 10 min. This process destroys most vegetative cells
and allows the spores to predominate. It has been shown to be
useful for the recovery and regeneration of solvent-producing
clostridia, such as C. acetobutylicum and C. beijerinckii.
Methods for detection and enumeration of C. perfringens
are found in a separate article. Although not as fastidious as
C. perfringens, the nutritional growth needs of C. botulinum are
complex and include a number of amino acids, B vitamins,
and minerals. Routinely, C. botulinum is cultivated in brain–
heart infusion or cooked meat medium. Although many
foods satisfy the nutritional requirements for growth, not all
provide anaerobic conditions. Growth in foods can be
restricted if the product is of low pH, has low aw, and has
a high concentration of salt or an inhibitory concentration of
a preservative, such as sodium nitrite. A food may contain
viable cells of C. botulinum, and yet it may not cause disease.
For this reason, the focus is primarily on detection of the
neurotoxin (see section Detection of C. botulinum Neuro-
toxins). Because of the heat lability of C. botulinum neurotoxin,
however, processed foods should be examined for the pres-
ence of viable cells as well as toxin.
The detection of viable C. botulinum typically involves
enrichment. Cooked meat medium or trypticase–peptone–
glucose–yeast extract (TPGY) is inoculated with 1–2 g solid or
1–2 ml liquid food and incubated. If the organism is suspected
of being a nonproteolytic strain, TPGY containing trypsin
should be used. After 7 days incubation, the culture is exam-
ined for gas production, turbidity, and digestion of
448 CLOSTRIDIUM j Introduction
meat particles. The culture also is examined microscopically.
A typical cell shows distention of the mother cell sporangium
due to the presence of the spore, which results in a bulging or
swollen appearance. If enrichment results in no growth after
7 days, the sample may be incubated for an additional 10 days
to detect injured cells or spores. Pure cultures of C. botulinum
are isolated by pretreatment of the sample with either absolute
alcohol or heat treatment (typically 80  C for 10 min). Heat- or
ethanol-treated cultures may be streaked on to anaerobic egg
yolk agar to obtain distinct and separate colonies. The selection
of typical C. botulinum colonies involves using a sterile transfer
loop to inoculate each isolated colony into TPGY or cooked
meat medium broth. Cultures are incubated for 7 days as
described and tested for toxin production. Repeated serial
transfer through enrichment media may help to increase the
cell numbers enough to permit pure colony isolation.
C. botulinum and C. perfringens are particularly important
species in the food industry because of their ability to produce
heat-stable spores and their ability to grow rapidly under
anaerobic conditions. Although normally producing only
a mild form of food poisoning, C. perfringens is of particular
concern to the food service industry in those cases in which
food is prepared in advance, reheated, and held on steam
tables. It is primarily problematic because of its ubiquitous
nature and rapid growth rate given appropriate nutritional and
environmental conditions. Because of the devastating nature of
botulism foodborne illness, minimum heating times for
ensuring the safety of canned foods have been developed with
the C. botulinum microorganism in mind.
Simple flow-chart based approaches for the identification
of Clostridium species are available as part of the National
Standard Methods Working Group (see http://www.hpa-
standardmethods.org.uk/wg_bacteriology.asp).
See also: Clostridium: Clostridium perfringens; Detection of
Enterotoxin of Clostridium perfringens; Clostridium : Clostridium
acetobutylicum; Clostridium: Clostridium tyrobutyricum;
Clostridium: Clostridium botulinum; Clostridium: Detection of
Neurotoxins of Clostridium botulinum; Bacterial Endospores;
Biochemical and Modern Identification Techniques: Food-
Poisoning Microorganisms; Detection of Enterotoxin of
Clostridium perfringens.
Further Reading
Andreesen, J.R., Bahl, H., Gottschalk, G., 1989. Introduction to the physiology and
biochemistry of the genus Clostridium. In: Minton, N.P., Clarke, D.J. (Eds.),
Clostridia. Plenum Press, New York, p. 27.
Blaschek, H.P., White, B.A., 1995. Genetic systems development in the clostridia.
FEMS Microbiology Reviews 17, 349–356.
Cato, E.P., George, W.L., Finegold, S.M., 1986. Genus Clostridium. In: Sneath, P.H.A.,
Mair, N.S., Sharpe, M.E., Holt, J.G. (Eds.), Bergey’s Manual of Systematic
Bacteriology. Williams and Wilkins, Baltimore, p. 1141.
Cato, E.P., Stackebrandt, E., 1989. Taxonomy and phylogeny. In: Minton, N.P.,
Clarke, D.J. (Eds.), Clostridia. Plenum Press, New York, pp. 1–26.
Hauschild, A., 1989. Clostridium botulinum. In: Doyle, M.P. (Ed.), Foodborne Bacterial
Pathogens. Marcel Dekker, New York, p. 112.
Jay, J.M., 1996. Modern Food Microbiology, fifth ed. Chapman & Hall, New York,
p. 220.
Johnson, J.L., Chen, J.-S., 1995. Taxonomic relationships among strains of Clos-
tridium acetobutylicum and other phenotypically similar organisms. In: Durre, P.,
Minton, N.P., Papoutsakis, E.T., Woods, D.R. (Eds.), Solventogenic Clostridia.
FEMS Microbiology Reviews, vol. 17, pp. 233–240.
Kautter, D.A., Solomon, H.M., Rhodehamel, E.J., 1992. Bacteriological Analytical
Manual, seventh ed. AOAC International, Arlington, VA, p. 215.
Milne, C.B., Eddy, J.A., Raju, R., Ardekani, S., Kim, P.-J., Senger, R.S., Jin, Y.-S.,
Blaschek, H.P., Price, N.D., 2011. Metabolic network reconstruction and genome-
scale model of butanol-producing strain Clostridium beijerinckii NCIMB 8052.
BMC Systems Biology 5, 130.
Morris, J.G., 1993. History and future potential of the clostridia in biotechnology. In:
Woods, D.R. (Ed.), The Clostridia and Biotechnology. Butterworth-Heinemann,
Stoneham, MA, p. 1.
Steinhart, C.E., Doyle, M.E., Cochrane, B.A., 1996. Food Safety. Marcel Dekker,
New York, p. 404.
Sugiyama, H., 1990. In: Cliver, D.O. (Ed.), Foodborne Diseases. Academic Press,
San Diego, CA, p. 108.
Wang, Y., Li, X., Mao, Y., Blaschek, H.P., 2011. Single-nucleotide resolution analysis
of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-
Seq. BMC Genomics 12, 479.
Wrigley, D.M., 1994. In: Hui, Y.H., Gorham, J.R., Murrell, K.D., Cliver, D.O. (Eds.),
Foodborne Disease Handbook: Diseases Caused by Bacteria, vol. 1. Marcel Dekker,
New York, p. 97.