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70 Modern Industrial Microbiology and Biotechnology
4.7.1.1.2 Use of enzymes
Enzymes hydrolyzing starch used to be called collectively diastase. With increased
knowledge about them, they are now called amylases. Enzymatic hydrolysis has several
advantages over the use of acid: (a) since the pH for enzyme hydrolysis is about neutral,
there is no need for special vessels which must stand the high temperature, pressure, and
corrosion of acid hydrolysis; (b) enzymes are more specific and hence there are fewer side
reactions leading therefore to higher yields; (c) acid hydrolysis often yields salts which
may have to be removed constantly or periodically thereby increasing cost; (d) it is
possible to use higher concentrations of the substrates with enzymes than with acids
because of enzyme specificity, and reduced possibility of side reactions.
4.7.1.1.3 Enzymes involved in the hydrolysis of starch
Several enzymes are important in the hydrolysis of starch. They are divisible into six
groups.
(i) Enzymes that hydrolyse a – 1, 4 bonds and by-pass a – I, 6 bonding: The typical
example is a - amylase. This enzyme hydrolyses randomly the inner (1 ® 4) - a -
D - glucosidic bonds of amylose and amylopectin (Fig. 4.3). The cleavage can occur
anywhere as long as there are at least six glucose residues on one side and at least
three on the other side of the bond to be broken. The result is a mixture of branched
a - limit dextrins (i.e., fragments resistant to hydrolysis and contain the a - D (1 6)
linkage (Fig. 4.4) derived from amylopectin) and linear glucose residues especially
maltohexoses, maltoheptoses and maltotrioses. a - Amylases are found in virtually
every living cell and the property and substrate pattern of a - amylases vary
according to their source. Thus, animal a - amylases in saliva and pancreatic juice
completely hydrolyze starch to maltose and D-glucose. Among microbial a -
amylases some can withstand temperatures near 100°C.
(ii) Enzymes that hydrolyse the a – 1, 4 bonding, but cannot by-pass the a – 1,6 bonds: Beta
amylase: This was originally found only in plants but has now been isolated from
micro-organisms. Beta amylase hydrolyses alternate a – 1,4 bonds sequentially
from the non-reducing end (i.e., the end without a hydroxyl group at the C – 1
position) to yield maltose (Figs. 4.3 and 4.5). Beta amylase has different actions on
amylose and amylopectin, because it cannot by-pass the a – 1:6 – branch points in
amylopectin. Therefore, while amylose is completely hydrolyzed to maltose,
amylopectin is only hydrolyzed to within two or three glucose units of the a – 1.6 -
branch point to yield maltose and a ‘beta-limit’ dextrin which is the parent
amylopectin with the ends trimmed off. Debranching enzymes (see below) are able
to open up the a – 1:6 bonds and thus convert beta-limit dextrins to yield a mixture
of linear chains of varying lengths; beta amylase then hydrolyzes these linear
chains. Those chains with an odd number of glucose molecules are hydrolyzed to
maltose, and one glucose unit per chain. The even numbered residues are
completely hydrolyzed to maltose. In practice there is a very large population of
chains and hence one glucose residue is produced for every two chains present in
the original starch.
Industrial Media and the Nutrition of Industrial Organisms 71
Fig. 4.5 Pattern of Attack of Alpha Amylase and Beta-amylase on Amylose and Amylopectin
Respectively
o = glucose units joined by a-D-(1 ® 4) bonds
l = non-reducing ends of chains
— = point of attack by enzyme
72 Modern Industrial Microbiology and Biotechnology
(iii) Enzymes that hydrolyze (a —1, 4 and a — 1:6 bonds: The typical example of these
enzymes is amyloglucosidase or glucoamylase. This enzyme hydrolyzes a - D - (1
® 4) -D – glucosidic bonds from the non-reducing ends to yield D – glucose
molecules. When the sequential removal of glucose reaches the point of branching
in amylopectin, the hydrolysis continues on the (1 ® 6) bonding but more slowly
than on the (1 ® 4) bonding. Maltose is attacked only very slowly. The end product
is glucose.
(iv) De-branching enzymes: At least two de-branching enzymes are known: pullulanase
and iso-amylase.
Pullulanase: This is a de-branching enzyme which causes the hydrolysis of a — D
– (1 ® 6) linkages in amylopectin or in amylopectin previsouly attacked by alpha-
amylase. It does not attack a - D (1 ® 4) bonds. However, there must be at least two
glucose units in the group attached to the rest of the molecules through an a -D- (1
® 6) bonding.
Iso-amylase: This is also a de-branching enzyme but differs from pullulanase in that
three glucose units in the group must be attached to the rest of the molecules
through an a - D – (1 ® 6) bonding for it to function.
(v) Enzymes that preferentially attack a - 1, 4 linkages: Examples of this group are
glucosidases. The maltodextrins and maltose produced by other enzymes are
cleaved to glucose by a - glucosidases. They may however sometime attack
unaltered polysaccharides but only very slowly.
(vi) Enzymes which hydrolyze starch to non-reducing cyclic D-glucose polymers known as
cyclodextrins or Schardinger dextrins: Cyclic sugar residues are produced by Bacillus
macerans. They are not acted upon by most amylases although enzymes in
Takadiastase produced by Aspergillus oryzae can degrade the residues.
4.7.1.1.4 Industrial saccharification of starch by enzymes
In industry the extent of the conversion of starch to sugar is measured in terms of dextrose
equivalent (D.E.). This is a measure of the reducing sugar content, expressed in terms of
dextrose, determined under defined conditions involving Fehling’s solution. The D.E is
calculated as percentage of the total solids.
For the saccharification of starch in industry acid is being replaced more and more by
enzymes. Sometimes acid is used only initially and enzymes employed at a later stage.
Acid saccharification has a practical upper limit of 55 D.E. Beyond this, breakdown
products begin to accumulate. Furthermore, with acid hydrolysis reversion reactions
occur among the sugar produced. These two deficiencies are avoided when enzymes are
utilized. Besides, by selecting enzymes specific sugars can be produced.
Starch-splitting enzymes used in industry are produced in germinated seeds and by
micro-organisms. Barley malt is widely used for the saccharification of starch. It contains
large amounts of various enzymes notably b-amylase and a - glucosidase which further
split saccharides to glucose.
All the enzymes discussed above are produced by different micro-organisms and
many of these enzymes are available commercially. The most commonly encountered
organisms producing these enzymes are Bacillus spp, Streptomyeces spp, Aspergillus spp,
Penicillium spp, Mucor spp and Rhizopus spp.
Industrial Media and the Nutrition of Industrial Organisms 73
4.7.2 Cellulose, Hemi-celluloses and Lignin in Plant Materials
4.7.2.1 Cellulose
Cellulose is the most abundant organic matter on earth. Unfortunately it does not exist
pure in nature and even the purest natural form (that found in cotton fibres) contains
about 6% of other materials. Three major components, cellulose, hemi-cellulose and
lignin occur roughly in the ratio of 4:3:3 in wood. Before looking more closely at cellulose,
the other two major components of plant materials will be briefly discussed.
4.7.2.2 Hemicelluloses
These are an ill-defined group of carbohydrates whose main and common characteristic
is that they are soluble in, and hence can be extracted with, dilute alkali. They can then be
precipitated with acid and ethanol. They are very easily hydrolyzed by chemical or
biological means. The nature of the hemicellulose varies from one plant to another. In
cotton the hemicelluloses are pectic substances, which are polymers of galactose. In
wood, they consist of short (DP less than 200) branched heteropolymers of glucose,
xylose, galactose, mannose and arabinose as well as uronic acids of glucose and
galactose linked by 1 – 3, 1 – 6 and 1 – 4 glycosidic bonding.
4.7.2.3 Lignin
Lignin is a complex three-dimensional polymer formed from cyclic alcohols. (Fig. 4.6). It
is important because it protects cellulose from hydrolysis.
Cellulose is found in plant cell-walls which are held together by a porous material
known as middle lamella. In wood the middle lamella is heavily impregnated with lignin
which is highly resistant and thus protects the cell from attack by enzymes or acid.
4.7.2.4 Pretreatment of cellulose-containing materials
before saccharification
In order to expose lignocellulosics to attack, a number of physical and chemical methods
are in use, or are being studied, for altering the fine structure of cellulose and/or breaking
the lignin-carbohydrate complex.
Table 4.8 Various pretreatment methods used in lignocellulose substrate preparation
Pretreatment type Specific method
Mechanical Weathering and milling-ball, fitz, hammer, roller
Irradiation Gamma, electron beam, photooxidation
Thermal Autohydrolysis, steam explosion, hydrothermolysis,
boiling, pyrolysis, moist or dry heat expansion
Alkali Sodium hydroxide, ammonium hydroxide
Acids Sulfuric, hydrochloric, nitric, phosphoric, maleic
Oxidizing agents Peracetic acid, sodium hypochlorite, sodium chlorite,
hydrogen peroxide
Solvents Ethanol, butanol, phenol, ethylamine, acetone, ethylene glycol
Gases Ammonia, chlorine, nitrous oxide, ozone, sulfur dioxide
Biological Ligninolytic fungi
74 Modern Industrial Microbiology and Biotechnology
Fig. 4.6 Generalized Structure of Lignin
Chemical methods include the use of swelling agents such a NaOH, some amines,
concentrated H
2
SO
4
or HCI or proprietary cellulose solvents such as ‘cadoxen’ (tris
thylene-diamine cadmium hydroxide). These agents introduce water between or within
the cellulose crystals making subsequent hydrolysis, easier. Steam has also been used as
a swelling agent. The lignin may be removed by treatment with dilute H
2
SO
4
at high
temperature.
Physical methods of pretreatment include grinding, irradiation and simply heating
the wood.
4.7.2.5 Hydrolysis of cellulose
Following pretreatment, wood may be hydrolyzed with dilute HCI, H
2
SO
4
or sulfites of
calcium, magnesium or sodium under high temperature and pressure as described for
sulfite liquor production in paper manufacture see section 4. above). When, however, the
aim is to hydrolyze wood to sugars, the treatment is continued for longer than is done for
paper manufacture.
A lot of experimental work has been done recently on the possible use of cellulolytic
enzymes for digesting cellulose. The advantage of the use of enzymes rather than harsh
chemicals methods have been discussed already. Fungi have been the main source of
cellulolytic enzymes. Trichoderma viride and T. koningii have been the most efficient
cellulase producers. Penicillicum funiculosum and Fusarium solani have also been shown
to possess equally potent cellulases. Cellulase has been resolved into at least three
components: C
1
, C
x
, and b-glucosidases. The C
1
component attacks crystalline cellulose
and loosens the cellulose chain, after which the other enzymes can attack cellulose. The
Industrial Media and the Nutrition of Industrial Organisms 75
C
x
enzymes are b - (1 ® 4) glucanases and hydrolyse soluble derivatives of cellulose or
swoollen or partially degraded cellulose. Their attack on the cellulose molecule is
random and cellobiose (2-sugar) and cellotroise (3-sugar) are the major products of their
actions. There is evidence that the enzymes may also act by removing successive glucose
units from the end of a cellulose molecule. b-glucosidases hydrolyze cellobiose and short-
chain oligo-saccharides derived from cellulose to glucose, but do not attack cellulose.
They are able to attack cellobiose and cellotriose rapidly. Many organisms described in
the literature as ‘cellulolytic’ produce only C
x
and b-glucosidases because they were
isolated initially using partially degraded cellulose. The four organisms mentioned
above produce all three members of the complex.
4.7.2.4.1 Molecular structure of cellulose
Cellulose is a linear polymer of D-glucose linked in the Beta-1, 4 glucosidic bondage. The
bonding is theoretically as vulnerable to hydrolysis as the one in starch. However,
cellulose – containing materials such as wood are difficult to hydrolyze because of (a) the
secondary and tertiary arrangement of cellulose molecules which confers a high
crystallinity on them and (b) the presence of lignin.
The degree of polymerization (D. P.) of cellulose molecule is variable, but ranges from
about 500 in wood pulp to about 10,000 in native cellulose.
When cellulose is hydrolyzed
with acid, a portion known as the amorphous portion which makes up 15% is easily and
quickly hydrolyzed leaving a highly crystalline residue (85%) whose DP is constant at
100-200. The crystalline portion occurs as small rod-like particles which can be
hydrolyzed only with strong acid. (Fig. 4.7)
A = Original cellulose fibril
B = Initial attack on amorphous region
C = Residue crystalline region
D = Attack on crystalline region
Fig. 4.7 Diagram Illustrating Breakdown of Crystalline Cellulose
5
76 Modern Industrial Microbiology and Biotechnology
SUGGESTED READINGS
Barnes, A.C. 1974. The Sugar cane. Wiley, New York, USA.
Dahod, S.K. 1999. Raw Materials Selection and Medium Development for Industrial
Fermentation Processes. In: Manual of Industrial Microbiology and Biotechnology. A. L.
Demain and J. E. Davies (eds) American Society for Microbiology Press. 2
nd
Ed, Washington
DC.
Demain, A.L. 1998. Induction of microbial secondary Metabolism International Microbiology 1,
259–264.
Flickinger, M.C., Drew, S.W. (eds) 1999. Encyclopedia of Bioprocess Technology - Fermentation,
Biocatalysis, and Bioseparation, Vol 1-5. John Wiley, New York.
Ward, W.P., Singh, A. 2004. Bioethanol Technology: Developments and Perspectives Advance in
Applied Microbiology, 51, 53 – 80.
Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products %%
5.1 THE NATURE OF METABOLIC PATHWAYS
In order to be able to manipulate microorganisms to produce maximally materials of
economic importance to humans, but at minimal costs, it is important that the physiology
of the organisms be understood as much as is possible. In this chapter relevant elements
of the physiology of industrial organisms will be discussed.
A yeast cell will divide and produce CO
2
under aerobic conditions if offered a solution
of glucose and ammonium salts. The increase in cell number resulting from the growth
and the bubbling of CO
2
are only external evidence of a vast number of chemical reactions
going on within the cell. The yeast cell on absorbing the glucose has to produce various
proteins which will form enzymes necessary to catalyze the various reactions concerned
with the manufacture of proteins, carbohydrates, lipids, and other components of the cell
as well as vitamins which will form coenzymes. A vast array of enzymes are produced as
the glucose and ammonium initially supplied are converted from one compound into
another or metabolized. The series of chemical reactions involved in converting a chemical (or a
metabolite) in the organism into a final product is known as a metabolic pathway. When the
reactions lead to the formation of a more complex substance, that particular form of
metabolism is known as anabolism and the pathway an anabolic pathway. When the
series of reactions lead to less complex compounds the metabolism is described as
catabolism. The compounds involved in a metabolic pathway are called intermediates and
the final product is known as the end-product (see Fig. 5.1).
Catabolic reactions have been mostly studied with glucose. Four pathways of glucose
breakdown to pyruvic acid (or glycolysis) are currently recognized. They will be
discussed later. Catabolic reactions often furnish energy in the form of ATP and
other high energy compounds, which are used for biosynthetic reactions. A second
function of catabolic reactions is to provide the carbon skeleton for biosynthesis.
Anabolic reactions lead to the formation of larger molecules some of which are
constituents of the cell.
Metabolic Pathways for the
Biosynthesis of Industrial
Microbiology Products
5
+0)26-4
%& Modern Industrial Microbiology and Biotechnology
Although anabolism and catabolism are distinct phenomena some pathways have
elements of both kinds Metabolic intermediates which are derived from catabolism and
which are also available for anabolism are known as amphibolic intermediates.
Methods for the study of metallic pathways are well reviewed in texts on microbial
physiology and will therefore not be discussed here.
5.2 INDUSTRIAL MICROBIOLOGICAL PRODUCTS AS
PRIMARY AND SECONDARY METABOLITES
Products of industrial microorganisms may be divided into two broad groups, those
which result from primary metabolism and others which derive from secondary
metabolism. The line between the two is not always clear cut, but the distinction is useful
in discussing industrial products.
5.2.1 Products of Primary Metabolism
Primary metabolism is the inter-related group of reactions within a microorganism
which are associated with growth and the maintenance of life. Primary metabolism is
essentially the same in all living things and is concerned with the release of energy, and
Fig. 5.1 Metabolism: Relationship between Anabolism and Catabolism in a Cell
Metabolic Pathways for the Biosynthesis of Industrial Microbiology Products %'
the synthesis of important macromolecules such as proteins, nucleic acids and other cell
constituents. When primary metabolism is stopped the organism dies.
Products of primary metabolism are associated with growth and their maximum
production occurs in the logarithmic phase of growth in a batch culture. Primary
catabolic products include ethanol, lactic acid, and butanol while anabolic products
include amino-acids, enzymes and nucleic acids. Single-cell proteins and yeasts would
also be regarded as primary products (Table 5.1)
Table 5.1 Some industrial products resulting from primary metabolism
Anabolic Products Catabolic Products
1. Enzymes 1. Ethanol and ethanol-containing products, e.g. wines
2. Amino acids 2. Butanol
3. Vitamins 3. Acetone
4. Polysaccharides 4. Lactic acid
5. Yeast cells 5. Acetic acid (vinegar)
6. Single cell protein
7. Nucleic acids
8. Citric acid
5.2.2 Products of Secondary Metabolism
In contrast to primary metabolism which is associated with the growth of the cell and the
continued existence of the organism, secondary metabolism, which was first observed in
higher plants, has the following characteristics
(i) Secondary metabolism has no apparent
function in the organism. The organism continues to exist if secondary metabolism is
blocked by a suitable biochemical means. On the other hand it would die if primary
metabolism were stopped. (ii) Secondary metabolites are produced in response to a
restriction in nutrients. They are therefore produced after the growth phase, at the end of
the logarithmic phase of growth and in the stationary phase (in a batch culture). They can
be more precisely controlled in a continuous culture. (iii) Secondary metabolism appears
to be restricted to some species of plants and microorganisms (and in a few cases to
animals). The products of secondary metabolism also appear to be characteristic of the
species. Both of these observations could, however, be due to the inadequacy of current
methods of recognizing secondary metabolites. (iv) Secondary metabolites usually have
‘bizarre’ and unusual chemical structures and several closely related metabolites may be
produced by the same organism in wild-type strains. This latter observation indicates the
existence of a variety of alternate and closely-related pathways. (v) The ability to produce
a particular secondary metabolite, especially in industrially important strains is easily
lost. This phenomenon is known as strain degeneration. (vi) Owing to the ease of the loss
of the ability to synthesize secondary metabolites, particularly when treated with acri-
dine dyes, exposure to high temperature or other treatments known to induce plasmid
loss (Chapter 5) secondary metabolite production is believed to be controlled by plasmids
(at least in some cases) rather than by the organism’s chromosomes. A confirmation of the
possible role of plasmids in the control of secondary metabolites is shown in the case of
leupetin, in which the loss of the metabolite following irradiation can be reversed by