Gerardi M.H. (ed.) The Microbiology of Anaerobic Digesters

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Substrates and Products

In chemical reactions there are reactants and products (Equation 8.1). During chem-

ical reactions, reactants (chemical compounds) undergo change and often release

energy (heat) to the environment. The changes that occur to the reactants result in

the formation of products (new chemical compounds). Often, a catalyst may be

involved in a chemical reaction. The catalyst accelerates the rate of the chemical

reaction and may be changed or consumed.

reactants — catalyst Æ products (8.1)

Chemical reactions that occur inside bacterial cells are known as biochemical

reactions. In biochemical reactions, reactants or substrates undergo change as bac-

terial cells degrade them. As the substrates are degraded, energy is released and

new compounds (products) are formed (Equation 8.2).

substrates Æ products (8.2)

Some of the energy released by the substrates is captured by the bacterial cells

and stored in high-energy phosphate bonds for use in cellular activity. Energy that

is not captured by the bacterial cells is lost to the environment as heat. New bacte-

rial cells and carbon dioxide are products of biochemical reactions that involve

organic compounds (Equation 8.3).

substrates (organic compounds) Æ bacterial cells + carbon dioxide (8.3)

Catalysts are involved in biochemical reactions. These catalysts are known as

enzymes. Enzymes are large proteinaceous molecules that greatly accelerate the

The Microbiology of Anaerobic Digesters, by Michael H. Gerardi

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rate of biochemical reactions. However, enzymes, unlike chemical catalysts, are not

altered or consumed during the reaction (Equation 8.4).

substrates — bacteria and bacterial enzymes Æ

bacterial cells + carbon dioxide (8.4)

During some biochemical reactions, intermediate products or “intermediates” are

formed (Equation 8.5). Intermediates usually are short-lived, that is, they do not

accumulate. However, speciﬁc environmental or operational conditions such as a

change in pH or temperature may permit the accumulation of intermediates. The

presence of some intermediates may result in operation problems in an anaerobic

digester.

substrates — intermediates Æ bacterial cells + carbon dioxide (8.5)

Initial substrates for bacteria in municipal anaerobic digesters include carbohy-

drates, lipids, and proteins. These substrates are found as particulates such as the

carbohydrate cellulose and as colloids such as proteins.

The degradation process or digestion of solids within an anaerobic digester con-

sists of three stages (Table 8.1). The ﬁrst stage is the hydrolysis of particulate and

colloidal wastes to soluble wastes in the form of organic acids and alcohols. The

second stage is the conversion of the organic acids and alcohols to acetate, carbon

dioxide, and hydrogen. The third stage is the production of gases, mostly methane,

and new bacterial cells or sludge from acetate and hydrogen. Because a great diver-

sity of bacteria are required in an anaerobic digester to perform hydrolysis, produce

acetate and hydrogen, and produce methane, the substrate feed to the digester

should contain a great diversity of wastes.

The net results of anaerobic digestion of solids are signiﬁcant decreases in

percent solids and percent volatile solids in digester sludge. The ﬁrst and second

stages of anaerobic digestion are achieved through the activities of facultative

anaerobes and anaerobes, whereas the third stage is achieved through the activity

of only anaerobes, the methane-forming bacteria.

Hydrolysis rates for particulate and colloidal wastes vary greatly according to

the waste to be degraded and the operational conditions at the time of hydrolysis.

Substrates hydrolyzed in the ﬁrst stage consist of carbohydrates, lipids, and

proteins. These substrates may be wasted to the digester from primary and second-

ary sludges.

62 SUBSTRATES AND PRODUCTS

TABLE 8.1 The Three Stages of Anaerobic Digestion of Solids

Stage Activity Enzymes Used

First Hydrolysis: Exoenzymes

Solubilization of particulate and colloidal wastes

Second Acid forming: Endoenzymes

Conversion of soluble organic acids and alcohols to

acetate, carbon dioxide, and hydrogen

Third Methanogenesis: Endoenzymes

Production of methane and carbon dioxide

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CARBOHYDRATES

Carbohydrates are synthesized in the green leaves of plants by the conversion of

carbon dioxide into glucose during photosynthesis. Carbohydrates are macromole-

cules or polymers that contain numerous monomers of sugars (Figure 8.1). The

range of lengths of the polymers or carbohydrates varies greatly.

Within the digester all carbohydrates are degraded inside the cell of facultative

anaerobes and anaerobes. Carbohydrates too large to enter the cell, that is, in an

insoluble or complex soluble form, must be hydrolyzed into smaller, soluble sugars

CARBOHYDRATES 63

3 - 7 carbon sugar

Mer or monomer, monosaccharide

2 mers or disaccharide

3 - 7 carbon sugar

Many mers or polysaccharide

Figure 8.1

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outside the cell through the use of exoenzymes (Figure 8.2). Once hydrolyzed, the

smaller, soluble sugars enter the cell, where they are degraded by endoenzymes.

The monomers of carbohydrates are simple sugars (Table 8.2). These sugars are

known as monosaccharides, and they contain three to seven carbon units. The

common generic formulae for monosaccharides are (CH

– (CH

. The major

monomers or monosaccharides in our diet are fructose and glucose. Although many

64 SUBSTRATES AND PRODUCTS

3 - 7 carbon sugar

Insoluble polysaccharide

Saccrolytic bacterium

Exoenzymes

3 - 7 carbon sugar

Endoenzymes

Figure 8.2 Large, complex, and insoluble carbohydrates or polysaccharides must be hydrolyzed by

sacchrolytic bacteria with the use of exoenzymes. Once solublized, the individual sugar units of the

polysaccharides can enter the bacterial cells and can be degraded by endoenzymes.

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monomers have identical chemical formulae, for example, glucose (C

) and

fructose (C

), they are structurally different (Figure 8.3).

When two monomers are linked together, disaccharides are formed (Table 8.3),

and when numerous monosaccharides are linked together, polysaccharides are

formed (Table 8.4). Major disaccharides in our diet are sucrose (table sugar) and

lactose (milk sugar). Disaccharides are carbohydrates composed of monosaccha-

rides linked by an acetal bond (–C–O–C–). Polysaccharides often are referred to as

complex carbohydrates. The largest digestible polysaccharide in our diet is starch.

This polysaccharide is found in grains such as wheat and rice, root vegetables such

as potatoes, and legumes such as beans and peas.

Although some sugars contain nitrogen and phosphorus, all sugars contain

carbon, hydrogen, and oxygen. The basic chemical formula for sugars is (CH

The word “carbohydrate” was used originally to describe glucose—the hydrate of

carbon (CH

O) or C

Monosaccharides are water soluble and are quickly and easily transported across

the cell wall and cell membrane into the bacterial cell. Disaccharides also are water

soluble but must be converted or hydrolyzed to monosaccharides before they can

enter the bacterial cell.

Polysaccharides are very large, complex insoluble sugars that have a high molec-

ular weight. These sugars require the presence of speciﬁc exoenzymes and, usually,

CARBOHYDRATES 65

TABLE 8.2 Common Monosaccharides or Simple

Sugars

Monosaccharide Carbon Units Formula

Deoxyribose 5 C

Glucose (dextrose) 6 C

Galactose 6 C

Fructose (levulose) 6 C

Ribose 5 C

Mannose 6 C

TABLE 8.3 Common Disaccharides

Disaccharide Composition of Monosaccharides

Lactose Galactose-Glucose

Maltose Glucose-Glucose

Sucrose Glucose-Fructose

Cellobiose Galactose-Galactose

TABLE 8.4 Common Polysaccharides

Agar

Amylopectin (starch)

Amylose (starch)

Cellulose

Fiber

Glycogen

Pectin

Vegetable gum

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several enzymatic steps to ensure their hydrolysis and degradation. Because of their

insoluble nature, complex structure, and need for speciﬁc exoenzymes and numer-

ous enzymatic steps, polysaccharides are degraded slowly.

When disaccharides and polysaccharides are hydrolyzed, monosaccharides are

released. When monosaccharides are degraded in an anaerobic digester, organic

acids and alcohols are produced (Table 8.5). Many of these compounds are further

degraded to volatile acids.

LIPIDS

Lipids are naturally occurring organic molecules found in animal and plant tissues.

Lipids do not dissolve in water, that is, lipids are extracted from animal and plant

tissues with nonpolar organic solvents such as ether.

66 SUBSTRATES AND PRODUCTS

Glucose

H OH

HHO

Fructose

Figure 8.3

TABLE 8.5 Acids and Alcohols Produced from

Monosaccharide Degradation

Genus End Product from Monosaccharide

Degradation

Clostridium Butanol, butyrate, isopropanol

Enterobacter Butanediol, ethanol, formate, lactate

Escherichia Acetate, ethanol, lactate, succinate

Lactobacillus Lactate

Streptococcus Lactate

Propionibacterium Acetate, propionate

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There are numerous groups of lipids. The lipids most often wasted to a munici-

pal anaerobic digester include fats and oils. These compounds are derived from glyc-

erol (Figure 8.4). Glycerol combined with three fatty acids produces a triglyceride

or fat.

Fatty acids are straight-chain carbon compounds containing a terminal carboxylic

acid group (–COOH) (Figure 8.5). There are approximately 40 naturally occurring

fatty acids (Table 8.6). Fatty acids without a double bond (=) between carbon units

are known as saturated fatty acids, for example, palmitic acid. Fatty acids with a

double bond between carbon units (–C=C–) are known as unsaturated fatty acids,

for example, oleic acid. Fatty acids with two or more double bonds between carbon

LIPIDS 67

Figure 8.4

(CH

)

CH=CH(CH

)

COOH

unsaturated oleic acid

(CH

)

COOH

saturated palmitic acid

Figure 8.5

TABLE 8.6 Some Common Fatty Acids

Name Carbon Units Saturated Unsaturated Number of Double Bonds

Lauric 12 X 0

Myristic 14 X 0

Palmitic 16 X 0

Stearic 18 X 0

Linoleic 18 X 2

Linolenic 18 X 3

Oleic 18 X 1

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units are known as polyunsaturated fatty acids, for example, linoleic acid. Palmitic

acid and stearic acid are the most abundant saturated fatty acids, and oleic acid and

linoleic acid are the most abundant unsaturated fatty acids.

Animal fats and vegetable fats or oils are the most abundant lipids in nature.

Examples of animal fats include butter and lard, and examples of vegetable oils

include corn, olive, peanut, soybean, and sunﬂower oils. All fats and oils have similar

chemical structures. They are triglycerides. The three fatty acids of a triglyceride are

not necessarily the same (Table 8.7).

Large and complex fatty acids, fats, and oils are hydrolyzed in an anaerobic

digester. The resulting small and simplistic molecules obtained from hydrolysis are

degraded further to organic acids.

In anaerobic digesters fats undergo degradation through two principal steps.

First, the fats are hydrolyzed to glycerol and fatty acids. Lipase enzymes are used

by bacteria to hydrolyze the fats. Glycerol is degraded, and the fatty acids released

through hydrolysis are degraded two carbon units at a time.

PROTEINS

The principal nitrogenous wastes in municipal sludges are proteins. Proteins are

complex, high molecular-weight compounds.These molecules have a relatively large

surface area and do not dissolve in wastewater or settle out of wastewater. Proteins

are made of amino acids that are either straight-chain (aliphatic) or ring-shaped

(cyclic) in structure (Figure 8.6). There are 20 different amino acids. Regardless of

their structure, all amino acids contain an amino group (–NH

) and a carboxyl group

(–COOH). The carboxyl group is the “acid” portion of the amino acid.

Amino acids are joined together by peptide bonds to form proteins (Figure 8.7).

Proteins consist of long chains of amino acids. Each protein has a unique composi-

tion and sequence of amino acids in its chain. The complex proteins formed from

peptide bonds cannot be transported into bacterial cells. The use of exoenzymes,

namely, proteases or peptidases, by bacteria to hydrolyze peptide bonds permits

the release of individual amino acids that are transported into bacteria cells (Figure

8.8). Once inside the cell, amino acids undergo additional degradation resulting in

the production of organic acids. Examples of amino acids fermented in anaerobic

digesters include alanine (Equation 8.6), arginine, glutamate, glycine, and lysine

(Table 8.8).

68 SUBSTRATES AND PRODUCTS

TABLE 8.7 Composition of Some Common Fats and Oils

Fat/Oil Animal Vegetable Principle Saturated Principle Unsaturated

Fat Oil Fatty Acid Fatty Acid

Butter X Palmitic Oleic

Lard X Palmitic Oleic

Corn X Palmitic Oleic

Olive X Palmitic, stearic Oleic

Peanut X Palmitic Oleic

Soybean X Palmitic Linoleic

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PROTEINS 69

COOH

Alanine (aliphatic amino acid)

CHCOOH

Phenylalanine (cyclic amino acid)

Figure 8.6

R-C-OH

N-R

amino acid-amino acid

peptide bond

Figure 8.7 Amino acids are joined together to form proteins. Amino acids are joined together through

the production of peptide bonds. The bonds are produced by joining the hydroxyl group (–OH) in the

carboxyl group (–COOH) of one amino acid with the amino group (–NH

) of other amino acid. When

the peptide bond is formed, water is produced.

TABLE 8.8 Amino Acids Commonly Fermented in

Anaerobic Digesters

Amino Acid Fermenting Bacteria

Alanine Clostridium propionicium

Arginine Clostridium spp.

Streptococcus spp.

Glutamate Clostridium tetanomorphium

Glycine Peptostreptococcus micros

Lysine Clostridium sticklandii

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