**2. The Clostridium**

Bacteria from the genus *Clostridium* share specific characteristics. Their capacity to form heat resistant spores and their intolerance to oxygen being the principals. Isolated from many

© 2012 Drouin and Lafrenière, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Drouin and Lafrenière, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

environments, they are generally considered as ubiquitous. Different species still require specific growth conditions; some are psychrophilic while other are mesophilic or even thermophilic. The genus also contains pathogenic species, like *Clostridium tetani*, *Clostridium botulinum* and *Clostridium perfringens*. Some species are recognized as plant endophyte and could fix atmospheric nitrogen (Minamisawa et al., 2004).

Cells from the genus *Clostridium* are defined as Gram-positive, endospore-forming rods and most species are obligate anaerobes with varying tolerance to oxygen (Pahlow et al., 2003). More than 100 species had been described in this genus, but recent advances in genetic phylogeny allow more specific classification of these organisms.

A group of clostria species had been recognized as important milk contaminant. Like most other *Clostridium*, even if these species are ubiquitous, they are responsible for a specific defect of some type of cheeses, called "late blowing" (see section below). Three species had been frequently detected in late blowing cheese samples: *Clostridium tyrobutyricum*, *Clostridium butyricum* and *Clostridium sporogenes* (Cocolin et al., 2004), with *C. tyrobutyricum*  being the dominant specie. Together, these species are called "butyric acid spores". Silage, a forage conservation technique, is frequently pointed as the principal source of butyric acid spores of ruminant feed. The specie *C. tyrobutyricum* is one of the most frequently isolated clostridial species in silage samples (Pahlow et al., 2003). *Clostridium* species commonly found in silage could be separated in three groups: proteolytic clostridia (group 1), *Clostridium butyricum* group (group 2), and *Clostridium tyrobutyricum* (group 3) (Pahlow et al., 2003). Group 1 and 2 clostridia proliferate at pH generally over 5, while *C. tyrobutyricum* group will grow at lower pH, but rarely under pH of 4.5. The *C. butyricum* group includes *Clostridium beijerinckii* and *Clostridium acetobutylicum* and, like *C. tyrobutyricum*, ferment a wide range of carbohydrates to butyric acid and acetic acid.

## **2.1. Physiology and ecology of the** *Clostridium tyrubutyricum*

The different species of the genus *Clostridium* colonized a wide range of ecological niches but some species could be found only in very specific habitat. Soil is generally considered the habitat for most species, but since their metabolism is mostly related to organic matter degradation, soil mainly acts as a reservoir for the preservation of their spores. *C. tyrobutyricum* is present in agricultural soil but his habitat is not clearly defined (type strain ATCC 25755), even if it will proliferate under other conditions.

*C. tyrobutyricum* main fermentative metabolism is saccharolytic, allowing reduction of carbohydrates and lactate to butyrate. The butyric acid fermentation pathway is summarized by the following stoichiometric reactions:

I: lactic acid + acetic acid → butyric acid + CO2

II: lactic acid → acetic acid + CO2 + 2H2

Lactic acid could come directly from the environment of the organism. Those pathways involve condensation of two pyruvate molecules, derived either from glucose or lactate (Figure 1).

could fix atmospheric nitrogen (Minamisawa et al., 2004).

phylogeny allow more specific classification of these organisms.

wide range of carbohydrates to butyric acid and acetic acid.

**2.1. Physiology and ecology of the** *Clostridium tyrubutyricum*

strain ATCC 25755), even if it will proliferate under other conditions.

summarized by the following stoichiometric reactions:

(Figure 1).

environments, they are generally considered as ubiquitous. Different species still require specific growth conditions; some are psychrophilic while other are mesophilic or even thermophilic. The genus also contains pathogenic species, like *Clostridium tetani*, *Clostridium botulinum* and *Clostridium perfringens*. Some species are recognized as plant endophyte and

Cells from the genus *Clostridium* are defined as Gram-positive, endospore-forming rods and most species are obligate anaerobes with varying tolerance to oxygen (Pahlow et al., 2003). More than 100 species had been described in this genus, but recent advances in genetic

A group of clostria species had been recognized as important milk contaminant. Like most other *Clostridium*, even if these species are ubiquitous, they are responsible for a specific defect of some type of cheeses, called "late blowing" (see section below). Three species had been frequently detected in late blowing cheese samples: *Clostridium tyrobutyricum*, *Clostridium butyricum* and *Clostridium sporogenes* (Cocolin et al., 2004), with *C. tyrobutyricum*  being the dominant specie. Together, these species are called "butyric acid spores". Silage, a forage conservation technique, is frequently pointed as the principal source of butyric acid spores of ruminant feed. The specie *C. tyrobutyricum* is one of the most frequently isolated clostridial species in silage samples (Pahlow et al., 2003). *Clostridium* species commonly found in silage could be separated in three groups: proteolytic clostridia (group 1), *Clostridium butyricum* group (group 2), and *Clostridium tyrobutyricum* (group 3) (Pahlow et al., 2003). Group 1 and 2 clostridia proliferate at pH generally over 5, while *C. tyrobutyricum* group will grow at lower pH, but rarely under pH of 4.5. The *C. butyricum* group includes *Clostridium beijerinckii* and *Clostridium acetobutylicum* and, like *C. tyrobutyricum*, ferment a

The different species of the genus *Clostridium* colonized a wide range of ecological niches but some species could be found only in very specific habitat. Soil is generally considered the habitat for most species, but since their metabolism is mostly related to organic matter degradation, soil mainly acts as a reservoir for the preservation of their spores. *C. tyrobutyricum* is present in agricultural soil but his habitat is not clearly defined (type

*C. tyrobutyricum* main fermentative metabolism is saccharolytic, allowing reduction of carbohydrates and lactate to butyrate. The butyric acid fermentation pathway is

I: lactic acid + acetic acid → butyric acid + CO2

II: lactic acid → acetic acid + CO2 + 2H2 Lactic acid could come directly from the environment of the organism. Those pathways involve condensation of two pyruvate molecules, derived either from glucose or lactate

**Figure 1.** Metabolic pathway of glucose fermentation by *Clostridium tyrobutyricum* (adapted from Zhu and Yang (2004) and Rooke and Hatfield (2003).

Soil and decaying plants, including older plant parts in close contact with soil is the natural habitat of *C. tyrobutyricum* (Ercolani, 1997). In forage stands, moist condition which favour microbial development and accumulation of dead leaves near the ground could produce conditions typical for the germination of clostridial spores. These conditions include low oxygen concentration, adequate humidity and presence of specific germination elicitors. For most clostridial species, molecules acting to promote germination include glucose, amino acids, organic acids and/or chelating molecule. For *C. tyrobutyricum*, acetate and ammonium are the principal germination compounds (Bergère, 1969). These two compounds are often present in decaying plant material because acetate is present following reduction of carbohydrates and deamination of amino acids. Recent work aim to understand clostridia development in cheese by scanning electron microscopy reported that L-alanine in conjunction with L-lactate was the most potent inducer of clostridial spore germination (Bassi et al., 2009).

When feed is contaminated by clostridial spores and ingested by the animal, spores could migrate through the rumen and concentrate in relation to total digesta volume. Digestion

processes contribute to concentrate the number of spores, which could be quite high in cow manure. However, the enteric environment does not generally provide an adequate environment for the germination of spores of these species. It only plays a role in population diversity and transport of the organism to other environments.

Clostridial cells usually stop growing in presence of O2, but growth resumes when O2 concentration is under the physiological limit of the species. Oxygen sensibility depends on the metabolic level at time of exposure. Vegetative *Clostridium* cells could tolerate low oxygen concentration for short time period. Oxygen tolerance had not been measured for every specie, but some species could tolerate concentration as high as 3% O2. Acidic conditions as measured by pH and osmotic pressure as measured by Aw are also physical conditions that limit growth of *Clostridium* species. In fermenters, *C. tyrobutyricum* was able to tolerate pH as low as 4.5 (Zhu & Yang, 2004) suggesting that it could grow in silage environment under a wide range of physico-chemical conditions. Other species of *Clostridium* could be present in the soil and decaying materials like small rodent or bird corpses and manure. *Clostridium tetanii* is a good example of clostridia species generally recognize as present in soil. *Clostridium sporogenes* could be found in soil and manure. Other highly pathogenic species could also be present in manure, *Clostridium perfringens* and *Clostridium botulinum* are some of them.

Many clostridial species are specialized and need specific conditions to grow. *Clostridium cellulolyticum* is a mesophilic (optimum growth temperature between 25 and 40oC) specie that is specialized in cellulose catabolism in composting process. Another example of a specialized specie is *Clostridium thermolyticum*, able to degrade cellulose to ethanol under thermophilic conditions (optimum growth temperature between 40 and 70oC) and so, use in industrial processes. *Clostridium difficile* is a specie mainly found in the intestinal tract of warm blood animal, like human, and may cause mortality after severe disturbance of intestinal microflora in hospital environment.

## **2.2. Diversity of Clostridium at the farm level**

On farm, many different environments exist thus leading to diversity of clostridial species. Recent advances in molecular diversity techniques were applied to clostridial populations in order to follow distribution of different species. Diversity studies were performed in different environments of environment like soil, milk, plant surfaces, landfills, water treatment plants, and biogas fermenter (Herman et al., 1995; Julien et al., 2008; Klijn et al., 1995; Knabel et al., 1997; Van Dyke & McCarthy, 2002). For most of these studies, PCR primer sets designed specifically to amplify *Clostridium* species related to Cluster I (Collins et al., 1994) were used. One of these studies reported diversity of clostridia species in four different environments on four milk farms in Quebec (Canada) from soil to the raw milk (Figure 2) (Julien et al., 2008). Diversity patterns obtained following PCR-DGGE in fresh forage and stored feed samples are distinct from soil and milk samples. Operational taxonomic unit, (representing isolated band from the diversity pattern of a sample), related to *C. tyrobutyricum* (32, 33, 35 and 38) are present in all environments, but their number of occurrence ratio is higher in stored feed. Soil diversity pattern shows less specificity in diversity, while milk clostridial diversity seem to be more related to contamination by feces since OTU related to *Clostridium disporicum* showed high occurrences. *C. disporicum* isolates are often present in swine manure and manure biofilm (Leung & Topp, 2001).

378 Milk Production – An Up-to-Date Overview of Animal Nutrition, Management and Health

diversity and transport of the organism to other environments.

*Clostridium botulinum* are some of them.

intestinal microflora in hospital environment.

**2.2. Diversity of Clostridium at the farm level** 

processes contribute to concentrate the number of spores, which could be quite high in cow manure. However, the enteric environment does not generally provide an adequate environment for the germination of spores of these species. It only plays a role in population

Clostridial cells usually stop growing in presence of O2, but growth resumes when O2 concentration is under the physiological limit of the species. Oxygen sensibility depends on the metabolic level at time of exposure. Vegetative *Clostridium* cells could tolerate low oxygen concentration for short time period. Oxygen tolerance had not been measured for every specie, but some species could tolerate concentration as high as 3% O2. Acidic conditions as measured by pH and osmotic pressure as measured by Aw are also physical conditions that limit growth of *Clostridium* species. In fermenters, *C. tyrobutyricum* was able to tolerate pH as low as 4.5 (Zhu & Yang, 2004) suggesting that it could grow in silage environment under a wide range of physico-chemical conditions. Other species of *Clostridium* could be present in the soil and decaying materials like small rodent or bird corpses and manure. *Clostridium tetanii* is a good example of clostridia species generally recognize as present in soil. *Clostridium sporogenes* could be found in soil and manure. Other highly pathogenic species could also be present in manure, *Clostridium perfringens* and

Many clostridial species are specialized and need specific conditions to grow. *Clostridium cellulolyticum* is a mesophilic (optimum growth temperature between 25 and 40oC) specie that is specialized in cellulose catabolism in composting process. Another example of a specialized specie is *Clostridium thermolyticum*, able to degrade cellulose to ethanol under thermophilic conditions (optimum growth temperature between 40 and 70oC) and so, use in industrial processes. *Clostridium difficile* is a specie mainly found in the intestinal tract of warm blood animal, like human, and may cause mortality after severe disturbance of

On farm, many different environments exist thus leading to diversity of clostridial species. Recent advances in molecular diversity techniques were applied to clostridial populations in order to follow distribution of different species. Diversity studies were performed in different environments of environment like soil, milk, plant surfaces, landfills, water treatment plants, and biogas fermenter (Herman et al., 1995; Julien et al., 2008; Klijn et al., 1995; Knabel et al., 1997; Van Dyke & McCarthy, 2002). For most of these studies, PCR primer sets designed specifically to amplify *Clostridium* species related to Cluster I (Collins et al., 1994) were used. One of these studies reported diversity of clostridia species in four different environments on four milk farms in Quebec (Canada) from soil to the raw milk (Figure 2) (Julien et al., 2008). Diversity patterns obtained following PCR-DGGE in fresh forage and stored feed samples are distinct from soil and milk samples. Operational taxonomic unit, (representing isolated band from the diversity pattern of a sample), related to *C. tyrobutyricum* (32, 33, 35 and 38) are present in all environments, but their number of

**Figure 2.** Diversity and frequency of occurrence of clostridia species from different farm environments determined by the molecular diversity technique PCR-denaturing gradient gel electrophoresis following amplification of Cluster 1 (Collins et al., 1994) associated species (Julien et al., 2008). Operational taxonomic unit 32, 33, 35 and 38 are similar to *C. tyrobutyricum* sequences in gene banks; OTU 38 is also closely related to *C. sporogenes*, a specie close to *C. tyrobutyricum*; OTU 42, 45, 46 and 47 are closely related to *Clostridium disporicum*
