Fermentation and industry 24346
Dr. Sabri M. Naser
Fermentative pathways of LAB and other Gram-positive bacteria
The Gram-positive bacteria are classified in two separate groups based on 16S rDNA
sequences: the Firmicutes and the Actinobacteria.
 The phylum Firmicutes contains unicellular gram positive Bacteria that have a
low mol % G + C content.
 The phylum Actinobacteria contains many filamentous, multicellular species
whose DNA has a high mol % G + C.
Figure 1. Phylogeny of Gram-positive bacteria
Diagram showing the two phyla of Gram-positive bacteria based on 16S rRNA analyses. Note that the unicellular, low
mol% G + C lactic acid bacteria and endospore-forming bacteria are found in the Firmicutes branch, whereas the
mycelial, high mol% G + C Actinobacteria form a separate branch.
1
Fermentation and industry 24346
Dr. Sabri M. Naser
1. PHYLUM: FIRMICUTES
The Firmicutes contains a large number of important physiological groups and species
that are discussed next.
Lactic Acid Bacteria (LAB)
Most of LAB have complex nutritional requirements. Many require amino acids and
vitamins, and some even need purines and pyrimidines to grow. Table 1 lists the major
genera of lactic acid bacteria as well as some of their common features.
Physiology and Metabolism
▪ Although all LAB carry out lactic acid fermentation, they can be separated into two
different groups based on the type of fermentation process.
1. The homofermentative bacteria carry out a simple fermentation in which lactic acid
is the sole product from sugar fermentations:
Glucose →2 lactic acid
The Embden-Meyerhof or glycolytic pathway is used in this process. The pyruvic
acid formed is reduced by the enzyme lactic acid dehydrogenase toproduce the
characteristic end product, lactic acid (Figure 2).
2. In contrast, the heterofermentative lactic acid bacteria produce ethanol and carbon
dioxide as well as lactic acid:
Glucose →Lactic acid+Ethanol+ CO2
- The heterofermentative pathway (Figure 3) lacks the key enzyme aldolase that is
present in the glycolytic pathway of the homofermentative lactics.
- Aldolase cleaves fructose-1, 6-diphosphate to form the two phosphorylated trioses
that ultimately lead to the production of two ATPs. Therefore, although
homofermenters obtain 2 moles of ATP for each mole of glucose fermented, the
heterofermenters obtain only 1 mole. This pathway involves an initial oxidation of
2
Fermentation and industry 24346
Dr. Sabri M. Naser
glucose to 6-phosphogluconic acid, which is decarboxylated to form CO2 and
ribulose 5-phosphate.
- The pentose formed is converted to:
(a) The three-carbon intermediate, glyceraldehyde-3-phosphate, which gives rise
to one molecule of ATP by substrate-level phosphorylation in the formation
of lactic acid.
(b) Acetyl phosphate, which is reduced to acetaldehyde, and then to ethanol.
- The only energy available to lactic acid bacteria is through ATP generated by
substrate-level phosphorylation. The resulting energy yield per mole of glucose is
very low compared to other bacteria. The homofermenters that produce 2 mol of
ATP per mole of glucose are twice as efficient as the heterofermenters. Although
very little energy is available for these bacteria by such fermentations, they have
been remarkably successful in establishing themselves in important niches in the
environment.
3
Fermentation and industry 24346
Dr. Sabri M. Naser
Figure 2. Homofermentation pathway
Pathway for dissimilation of glucose by homofermentative lactic acid bacteria. These bacteria use the
glycolytic pathway for formation of pyruvate and lactate dehydrogenase to produce lactate as the final end
product.
4
Fermentation and industry 24346
Dr. Sabri M. Naser
▪ The optical form of lactic acid produced by lactic acid bacteria varies in the different
genera. Some produce one stereoisomer, the D-form, which rotates light toward the right
(dextro rotary), whereas others produce the L-form (left or levo rotary). The reason for
these differences lies in the stereospecificities of the lactic dehydrogenase enzyme itself.
Some species produce enzymes that make both D- and L-forms, resulting in a racemic
mixture of the two.
▪ Lactic acid bacteria can grow in the presence of oxygen but are unable to use it
metabolically. They lack cytochrome enzymes (which have iron-containing heme groups
that they cannot synthesize) and an electron transport system with which to generate ATP
by electron transport phosphorylation. Lactic acid bacteria do have flavoproteins,
however, and when they are exposed to oxygen, they produce hydrogen peroxide that can
be toxic to the cells.
▪ It is interesting to note that most lactic acid bacteria are catalase negative; that is, they
are unable to make the enzyme catalase, which like cytochromes, are ironcontaining
heme-proteins that degrade peroxides in the following manner:
H2O2→2 H2O + O2
Those species lacking catalase have other enzymes, peroxidases that degrade hydrogen
peroxide through organic compound-mediated reductions. Reactive oxygen compounds,
such as hydrogen peroxide, can lead to damage of almost all cell components, including
nucleic acids, lipid membranes, and proteins. Enzymes, such as peroxidases, degrade the
toxic oxygen forms thereby providing a protective effect. Another toxic form of oxygen
is the superoxide anion, O2–, which is formed in various physical and biochemical ways
by single electron reductions of O2. The lactic acid bacteria as well as most aerobic and
facultative aerobic bacteria protect themselves from this strong oxidizing agent by
producing the enzyme superoxide dismutase, which decomposes superoxide as follows:
2 O2– + 2 H+ →O2 + H2O2
Note that hydrogen peroxide is formed by the enzyme.
The concerted action of the superoxide dismutase and either catalases or peroxidases
results in the destruction of these toxic-oxidizing agents.
5
Fermentation and industry 24346
Dr. Sabri M. Naser
Figure 3. Heterofermentation pathway
Pathway for dissimilation of glucose by heterofermentative lactic acid bacteria. These bacteria lack aldolase, a key
glycolytic enzyme, and therefore do not use the Embden-Meyerhof fermentation pathway.
6
Fermentation and industry 24346
Dr. Sabri M. Naser
Clostridium
▪ This genus of anaerobic spore formers is even more diverse than Bacillus and contains
some important environmental species as well as human pathogens.
▪ These bacteria are obligate anaerobes that lack cytochrome and an electron transport
system. Thus, they rely solely on the formation of ATP by substrate level
phosphorylations during fermentation of various carbon sources.
▪ The genus is metabolically diverse. Two species, C. cellobioparum and C.
thermocellum, carry out an anaerobic fermentation of cellulose to form the disaccharide
cellobiose, which is ultimately fermented to produce acetic and lactic acids, ethanol, H 2,
and CO2 as the major end products.
-
C. cellobioparum is found in the rumen of cattle and sheep and enables these
higher animals to utilize cellulose in their diet. Interestingly, this organism is
inhibited when too much H2 accumulates as a result of the fermentation.
-
These bacteria live in close association with other ruminant microbes, the
methanogenic bacteria, which remove the hydrogen by forming methane gas. The
removal of the hydrogen gas lowers its concentration to a sufficiently low level
that the formation of more hydrogen gas is favored. This interaction involving
two different groups is referred to as an interspecies hydrogen transfer and is an
example of synergism. C. thermocellum is common in decaying soils containing
cellulose.
▪ A major group of the clostridia ferment sugars and occasionally starch and pectin to
form butyric acid, acetic acid, CO2, and H2 as the principal end products.
- The pathway for this fermentation is shown in Figure 4. In this butyric acid
fermentation, glucose is fermented to pyruvic acid via the Embden-Meyerhof
pathway.
- The pyruvate is split into carbon dioxide and hydrogen gas in the formation of
acetyl-coenzyme A. Some of the acetyl-CoA is used for ATP generation in the
formation of acetic acid via acetyl-phosphate.
- Also, the acetyl-CoA can be condensed with another molecule of acetyl CoA to
form acetoacetyl-CoA, which is the precursor of butyric acid.
- These are the so-called acetone-butanol fermenters. Butanol is formed from butyrylCoA via butyrylaldehyde (see Figure 4). Acetone and isopropanol are formed from
acetoacetyl-CoA by decarboxylation and subsequent reduction, respectively. These
so-called butyric acid bacteria include Clostridium pasteurianum, which can fix
nitrogen, a property shared by several other species in this group.
- As the acids accumulate during butyric acid fermentation, some species, such as
Clostriduim acetobutylicum, begin to produce more neutral compounds, including
butanol and acetone. The acetone-butanol fermentation has been used
commercially. It was especially important during World War I because of the need
for acetone in munitions manufacture. In this regard, it is interesting to note that
recently clostridia have been shown to be degraders of munitions, such as the
explosive TNT (trinitrotoluene).
7
Fermentation and industry 24346
Dr. Sabri M. Naser
Figure 4. Butyric acid fermentation
In butyric acid fermentation, pyruvate formed by the Embden-Meyerhof pathway is broken down to CO2, H2, and
acetyl-CoA. The acetyl-CoA is converted to acetate or to acetoacetyl-CoA, the precursor of butyrate. Acetone-butanol
fermenters convert the acetoacetyl- CoA to butanol, acetone, and isopropanol.
8
Fermentation and industry 24346
Dr. Sabri M. Naser
▪ Many butyric acid species of Clostridium are proteolytic; that is, they carry out the
anaerobic hydrolysis of proteins resulting in amino acids. The amino acids can then be
fermented with the production of ATP.
-
Figure 5 shows an example of a typical fermentation for glutamic acid.
-
The end products of this particular fermentation are the same as the butyric acid
fermentation, except that ammonia is also formed.
9
Fermentation and industry 24346
Dr. Sabri M. Naser
Figure 5. Glutamic acid fermentation
Many clostridia ferment glutamate to ammonia, acetate, and butyrate.
10
Fermentation and industry 24346
Dr. Sabri M. Naser
2. PHYLUM: ACTINOBACTERIA
▪ The “true” Actinobacteria (also referred to as the “actinomycetes”) are mycelial
organisms; that is, the cells produce branches as well as filaments. Therefore, they have a
mycelial growth habit resembling that of the fungi.
▪ One of the first organisms studied in this group was the genus Actinomyces, after which
the group is named. Some species produce only a substrate mycelium in which the
growth is either within or on the surface of the agar or other growth medium.
▪ Almost all of the actinomycetes are nonmotile; however, some types produce flagellated
spore that permit dispersal in aquatic habitats. Many members of the Actinobacteria
produce multicellular filaments or mycelia.
Propionibacterium
- This genus is so named because they produce propionic acid as a principal product of
their fermentation.
- They are gram-positive aerotolerant fermentative bacteria that are found in two different
habitats.
- One habitat occupied by the classic type of Propionibacterium is the intestinal tract of
animals.
- An allied habitat is cheese. Indeed, the characteristics of Swiss (Ementhaler) cheese are
due to the growth of propionibacteria.
- These organisms take the lactic acid formed by the lactic acid bacteria in cheese
fermentation and further metabolize it to propionic and acetic acids as well as CO2
(Figure 6).
- These reactions are more complex, comprising a series of steps beginning with the
oxidation of lactic acid to pyruvate and then transformation of the pyruvate (Figure 7).
The gas production the likely original source of these organisms in the cheese-making
industry.
- Sugars are also fermented by propionibacteria. They use the Embden-Meyerhof
pathway to produce pyruvate, which is then fermented as shown in Figure 7.
- The other major habitat of propionibacteria is the skin of mammals. One species,
Propionibacterium acnes, is found on the skin of all humans. It grows in the sebaceous
gland (not sweat glands), where it produces propionic acid in abundance. It ferments the
lactic acid produced by Staphylococcus epidermidis to form propionic and acetic acid as
previously discussed. Because propionic and acetic acids are volatile fatty acids with
distinctive smells, they give animals, including humans, a characteristic natural scent.
11
Fermentation and industry 24346
Dr. Sabri M. Naser
Figure 6. Propionic acid fermentation
Propionibacterium spp. ferment lactate to propionate and acetate. The route from pyruvate to propionate is shown in
Figure 7.
12
Fermentation and industry 24346
Dr. Sabri M. Naser
Figure 7. Propionibacterium sugar fermentation
In propionic acid fermentation, either sugars or lactate can serve as substrate.
13
Fermentation and industry 24346
Dr. Sabri M. Naser
Highly Branched and Mycelial Actinobacteria
- The more highly branched Actinobacteria are common soil organisms that are
prokaryotic counterparts of the fungi (Table 3).
- Some of them produce an aerial mycelium that has a fruiting structure bearing spores
called conidiospores. These are asexual spores, sometimes referred to as exospores to
differentiate them from endospores.
Actinomyces
- This genus, for which the entire group was named, is atypical of other mycelial
members, primarily because it is a group of anaerobic or facultative anaerobic bacteria.
- They ferment sugars such as glucose to produce formic, acetic, lactic, and succinic acids
and do not carry out aerobic respiration. Organic nitrogen compounds are required for
growth, and supplemental carbon dioxide greatly enhances it.
- Though they are mycelial, they do not produce an aerial mycelium. They grow in the
oral cavities of animals.
Important genera of branching and mycelial bacteria
14
Fermentation and industry 24346
Dr. Sabri M. Naser
Figure 8. Bifidobacterium fermentation
The bifidobacteria carry out an unusual lactic acid fermentation
Bifidobacterium
- Although Bifidobacterium is not a mycelial organism, its phylogeny places it as a close
relative of Actinomyces.
- This is a genus of anaerobic irregular rod-shaped bacteria that ferment sugars to acetic
and lactic acids (Figure 8).
- They are found primarily in the intestinal tracts of animals. One species, B. bifidus, is
commonly found in the intestines of humans that are breast fed and is therefore a pioneer
colonizer of the human intestinal tract. It is particularly well adapted to growing on
15
Fermentation and industry 24346
Dr. Sabri M. Naser
human breast milk, which contains an amino sugar disaccharide not found in cow’s milk.
This species is unusual in that it requires amino sugars for growth.
Streptomyces
- The foremost genus of the mycelial actinobacteria is Streptomyces. This is a diverse
group of soil bacteria that produce aerial as well as substrate mycelia.
- The hyphae, or filaments, of the aerial mycelia differentiate to form asexual
conidiospores. The spores of this genus are formed in chains at the tips of aerial hyphae..
- Members of this genus grow on simple inorganic media supplemented with a variety of
organic carbon sources, including glucose or glycerol; vitamins are not required. They
metabolize by aerobic respiration. In addition to simple organic carbon sources, some can
use polysaccharides such as pectin and chitin.
- This group is noted for the commercially important antibiotics they produce including
streptomycin, chloramphenicol, and tetracycline (Table 4). Approximately half of the
commercially produced antibiotics are derived from this genus. In addition, they are
sources of anticancer drugs used in chemotherapy. Although this is an extremely
important genus of soil microorganisms, little is known of their ecological roles.
- Other genera of mycelial actinomycetes are differentiated from this genus primarily by
their cell wall composition and the morphology of their conidiosporebearing structure.
Important antibiotics produced by Streptomyces species
Clostridium spp. ferment cellulose, sugars, amino acids, or purines. In the Stickland
fermentation, two amino acids are catabolized: one amino acid is fermented, the other is
oxidized. One species, Clostridium aceticum, is an acetogen that obtains energy from
oxidation of H2 and fixes CO2 via the acetyl-coenzyme A pathway. Clostridium
botulinum is the causative agent of botulism;
16
Fermentation and industry 24346
Dr. Sabri M. Naser
Clostridium tetani is the causative agent of tetanus. The mycoplasmas, or Mollicutes,
lack a cell wall, but are close relatives of Clostridium—many species, such as
Mycoplasma pneumoniae, are pathogenic to humans. Micrococcus spp. differ from
Staphylococcus in that they are obligate aerobes that are common soil inhabitants and
have a higher mol % G + C content.
Propionibacterium is noted for its production of propionic acid from lactic acid; it is
responsible for Swiss cheese production. Bifidobacterium bifidus, which ferment sugars
to acetic and lactic acids, are part of the normal microbiota of breast-fed infants.
Corynebacterium is the formally named genus of the coryneform bacteria that are
common soil organisms. Corynebacterium diphtheriae lives as a parasite of the oral
cavities of humans—strains that cause diphtheria have received a piece of DNA from a
phage that encodes for the diphtheria protein exotoxin.
The actinomycetes are a group of branching filamentous organisms, some of which
produce both a substrate and an aerial mycelium. Mycobacterium spp are acid-fast and
produce mycolic acids; two species, M. tuberculosis and M. leprae (leprosy), are wellknown pathogens.
Streptomyces is a common soil actinomycete noted for its production of antibiotics such
as tetracyclines, chloramphenicol, streptomycin, and erythromycin. Streptomyces impart
an “earthy” smell to soil due to the production of geosmins.
17