Fermentation Is the Metabolic Action of Bacteria

In fermentative digestion, molecular substrates are broken down by the action of bacteria and other microorganisms. Enzymatic hydrolysis of large molecules is an essential part of fermentative digestion, just as it is for glandular digestion. The major difference between the two processes is that the enzymes of fermentative digestion are microbial in origin, rather than coming from the host animal. Other major differences between fermentative and glandular digestion involve the rate of reactions and the extent of alteration of the substrate molecules. In general, fermentative digestion is much slower than glandular digestion, and the substrates are altered to a much greater degree.

The Sites of Fermentative Digestion Must Be Conducive to Microbial Growth

Fermentative digestion occurs in specialized compartments that are positioned either before or after the stomach and small intestine. Fermentative compartments positioned before the stomach are called forestomachs and are most highly developed in the ruminants and camelids. The size and development of the forestomach fermentation compartments vary greatly among species; many species have distinct forestomachs that are less developed than those of ruminants. Some species, including the horse and rat, have no anatomically distinct forestomach; however, some fermentative digestion may occur in a nonglandular portion of the proximal stomach.

Fermentation compartments positioned distal to the small intestine are the cecum and colon, often collectively called the hindgut. As with the forestomach, great anatomical differences exist in the hindgut of various species. This variation can be so extensive that the cecum and colon may appear to be functionally different organs in different species; however, when the variations are evaluated critically, important similarities can be seen in hindgut function among species.

The forestomach and hindgut can support fermentative digestion because their pH, moisture, ionic strength, and oxidation-reduction conditions are maintained in a range compatible for the growth of suitable microbes. In addition, the flow of ingesta through these areas is comparatively slow, allowing microbes time to maintain their population size. The importance of these factors can be illustrated through comparison of the forestomach and colon to the stomach and small intestine. In the stomach, bacterial numbers are kept low by the acid pH, whereas in the small intestine, bacterial numbers are kept in check by the constant flushing action of ingesta and secretions. In contrast, the pH in the forestomach and large colon is close to neutral, and the flow rate is comparatively slow.

In general, the fermentative patterns of the hindgut appear to be similar to those of the forestomach, although forestomach fermentation, especially that of the rumen, appears to be the better studied of the two. The following discussion focuses on rumen digestion but includes comments on hindgut digestion. Digestion in the equine cecum and colon is discussed at the end of the chapter.

Cooperation and Interplay Among the Many Species of Microbes Give Rise to a Complex Ecosystem in the Forestomach and Hindgut

The digestive process in the rumen or colon involves the interplay among the many species of bacteria and other microbes. The ecosystem of fermentative digestion is extremely complex, with the waste products of one microbial species serving as substrate for another. For example, Ruminococcus albus and Bacteroides ruminicola appear to exist synergistically. R. albus digests cellulose (is cellulolytic) but cannot digest protein. B. ruminicola, on the other hand, can digest protein but cannot digest cellulose. When the microbes are grown together, cellulose digestion by R. albus provides hexoses for the energy needs of B. ruminicola, and protein digestion by B. ruminicola provides ammonia and branch-chain fatty acids for the growth needs of R. albus.

In addition to substrate needs, growth factor needs are also supplied synergistically within the rumen ecosystem. For example, B vitamins are necessary for the growth of several rumen microbes, but these nutrients are generally not necessary in ruminant diets. The synergistic effect of B vitamins results from cross-feeding between species of those microbes that produce various B vitamins and those microbes that require them.

Despite tremendous ecological complexity, however, the entire pattern of fermentation may be viewed as a holistic process, without consideration of the roles and interactions of individual microbial species. Fermentative digestion is examined here from this viewpoint, with the actions of the entire rumen biomass considered as an overall digestive process, irrespective of the specific needs and actions of individual microbial species.

Nutrients Other Than Cell Walls Are Also Subject to Fermentative Digestion

The fermentative digestion of plant cell-wall material and its importance to herbivore digestion are well known. In addition, however, essentially all protein and carbohydrate nutrients that can provide substrate for energy and growth in mammals can also support the similar needs of microbes. Therefore, almost all dietary protein and carbohydrate are potentially subject to fermentative digestion. This fact is especially important in ruminants, in which food is exposed to fermentative digestion in the forestomach before its arrival at sites of glandular digestion. This temporal arrangement leads to the fermentative digestion of many nutrients that would otherwise have been available to the animal through glandular digestion. Thus, forestomach fermentative digestion, which provides for the efficient use of plant cell walls, can potentially lead to the inefficient use of other nutrients because of microbial alteration.

Anaerobic Conditions in the Rumen Result in Metabolic Activities Leading to the Production of Volatile Fatty Acids

When carbohydrate material enters the rumen or colon, it is attacked by hydrolytic microbial enzymes. In the case of insoluble carbohydrates, attack requires the physical attachment of bacteria to the surface of the plant particle, with the enzymes themselves part of the surface coating of the bacteria. Enzymatic action liberates glucose, other monosaccharides, and short-chain polysaccharides into the fluid phase, outside the microbial cell bodies. Although free in solution, these products of microbial enzyme action do not become immediately available to the host animal; rather, they are quickly subjected to further metabolism by the microbial mass. Glucose and other sugars are absorbed into the cell bodies of the microbes.

Within the microbial cells, glucose enters the glycolytic, or Embden-Meyerhof, pathway. This is the same glycolytic pathway that exists in mammalian cells, and as in mammalian tissues, catabolism of glucose through this pathway yields two molecules of pyruvate for each molecule of glucose metabolized. In the process, two molecules of oxidized nicotinamide adenine dinucleotide (NAD) are reduced to NAD hydrogen (NADH), and two molecules of adenosine triphosphate (ATP) are formed from adenosine diphosphate (ADP). The potential energy represented by the ATP formed in this reaction is not directly available to the host animal but is the major source of energy for maintenance and growth of microbes.

If fermentative digestion were to occur under aerobic conditions, which it does not, the pyruvate produced by the glycolytic process would enter the citric acid (Krebs) cycle and would be metabolized to carbon dioxide and water, as occurs under the aerobic conditions in mammalian cells. Furthermore, in an aerobic system, the NADH produced would be oxidized in the cytochrome oxidase system with additional production of ATP and the regeneration of NAD. However, fermentative digestion is not an aerobic system; on the contrary, it proceeds in a reductive, highly anaerobic environment. Therefore a different mechanism must be provided for the oxidation of NADH and other reduced cofactors. If such a mechanism were not available, all the oxidized cofactors present would soon be reduced, and metabolism would come to a halt. Because no atmospheric oxygen is available, some other compound must serve as an “electron sink” for the oxidation of enzyme cofactors.

In fermentative digestion, pyruvate can act as an electron sink, being further reduced to provide for regeneration of NAD and the general removal of excess electrons, with an additional yield of ATP. Also, carbon dioxide can be reduced to methane, accepting electrons for the regeneration of NAD. Figure 31-1 illustrates the metabolic pathways of these reactions. These pathways lead to the major end products of the fermentative digestion of carbohydrate, the volatile fatty acids (VFAs). The primary VFAs are acetic acid, propionic acid, and butyric acid; the VFAs are often referred to as their dissociated ions: acetate, propionate, and butyrate, respectively. Other quantitatively minor but metabolically important VFAs are valeric acid, isovaleric acid, isobutyric acid, and 2-methylbutyric acid. Figure 31-2 shows the chemical structures of the VFAs.

Production of propionic acid from pyruvate results in the efficient regeneration of NAD with no net production of NADH. In fact, production of available oxygen by the randomizing branch of the propionic acid pathway leads to oxidation of excess NADH originating from the acetic or butyric acid pathways (see Figure 31-1). The production of acetic acid leads to the efficient generation of ATP but, in contrast to the production of propionic acid, does not result in the regeneration of NAD from NADH. In the acetic acid pathway, excess NADH is produced. In this case, NAD is regenerated by the formation of free hydrogen, which is subsequently used to reduce carbon dioxide to methane and water (see Figure 31-1, lower portion).

Thus a direct relationship exists between acetic acid production and methane production; as the amount of pyruvate entering the acetic acid pathway increases, there must be a concomitant rise in methane production. Likewise, a reciprocal relationship exists between methane production and propionic acid production; as pyruvate is diverted to propionic acid production, there is less need for methane synthesis. These relationships are shown in the stoichiometric equations of Box 31-2. These reactions do not, however, fully describe the flow of hydrogen, or reducing substances, in rumen or colonic metabolism. The chemical reactions of fermentation are extremely complex and interdependent, and NADH can donate its electrons to reactions other than those described in Box 31-2, such as the synthesis of microbial protein and the saturation of unsaturated fatty acids.

In the rumen, methane production is facilitated by methanogenic bacteria, such as Methanobacterium ruminantium. This fragile bacterium is sensitive to changing conditions in the rumen. When conditions are unfavorable for the survival of M. ruminantium, methane production is reduced, shifting the metabolic pathways toward propionic acid production. Some conditions that suppress methanogenic species are high levels of feed intake, use of finely ground or pelleted feeds, and high-grain or high-starch diets. Under these circumstances the rate of methane production is reduced, resulting in a lower rate of acetic acid production with a concomitant increase in the propionic acid production rate.

The proportional rates at which acetic acid, propionic acid, and butyric acid are produced are reflected in their relative concentrations in the rumen fluid. The relative concentrations of the VFAs have important nutritional and metabolic consequences, and although seldom measured for medical purposes, VFA concentrations are frequently reported in research literature. Typically, the ruminal acetic/propionic/butyric acid concentration ratio in ruminants ranges from 70 : 20 : 10 for animals eating high-forage diets to 60 : 30 : 10 for animals eating high-grain diets. One must remember that these values represent relative proportions and not absolute amounts. The total amount of VFA produced with a high-starch diet is usually much higher than that produced with a high-fiber diet, such that total acetic acid production may be higher with a high-starch diet than with a high-fiber diet, even though the acetic acid production relative to the other VFAs may be reduced. Figure 31-3 illustrates this principle.

Volatile Fatty Acids Are Important Energy Substrates for the Host Animal

One can appreciate the elegance and beauty of the symbiotic relationship represented by fermentative digestion by considering the metabolism of VFAs. These molecules are the end products, indeed, the waste products, of anaerobic microbial metabolism, just as carbon dioxide is the waste product of aerobic metabolism. If the VFAs were allowed to accumulate, they would suppress or alter the fermentative process by lowering the pH of the gut or forestomach. However, the host animal maintains conditions for fermentation both by buffering pH changes and by removing VFAs from the gut by absorption. The benefit derived by the host is from the chemical energy that is contained in the VFAs. These bacterial “waste products” represent spent compounds within the framework of the anaerobic fermentation system, but they still contain considerable energy that can be derived from aerobic metabolism. In ruminants and other large herbivores, the VFAs are the major energy fuels, to a large extent serving the role played by glucose in omnivorous monogastric animals. The metabolic fates of the VFAs are discussed further in Chapter 32.

Fermentative Digestion of Protein Results in the Deamination of a Large Portion of Amino Acids

To this point, the discussion of fermentative digestion has centered primarily on carbohydrates, but as previously mentioned, other energy-yielding substrates are subject to microbial attack as well. Proteins are particularly vulnerable because they are composed of carbon compounds that can be further reduced to provide energy for anaerobic microbes. As proteins enter fermentative areas of the gut, they are attacked by extracellular microbial proteases. The majority of these enzymes are “trypsin-like” endopeptidases that form short-chain peptides as end products. These peptides are formed extracellularly and are absorbed into the microbial cell bodies, much as glucose is formed from carbohydrate and then absorbed. Within the microbial cells, the peptides can be used to form microbial protein or can be further degraded for the production of energy through the VFA pathways (Figure 31-4).

To enter the VFA pathways, the individual amino acids are first deaminated to yield ammonia (NH₃) and a carbon skeleton. The carbon structures of many of the amino acids can fit directly into various steps of the pathways leading to the production of the three major VFAs. The three branch-chain amino acids (BCAAs) are exceptions, however, and lead to the production of branch-chain VFAs by the following reactions:

Valine+2H2O→Isobutyrate+NH3+CO2

Leucine+2H2O→Isovalerate+NH3+CO2

Isoleucine+2H2O→2-Methylbutyrate+NH3+CO2

These branch-chain VFAs are important growth factors for several species of bacteria, as described later.

Although many species of rumen microbes appear capable of using preformed amino acids for the synthesis of protein, several species cannot do so. These species must synthesize amino acids from ammonia and the various carbon metabolites of the VFA pathways. For synthesis of the BCAAs, the branch-chain VFAs are required. Among the microbial species that require ammonia and branch-chain fatty acids are some of the important cellulose-digesting bacteria.

When Protein and Energy Availability in the Forestomach Are Well Matched, Rapid Microbial Growth and Efficient Protein Utilization Result

Because a large part of preformed dietary protein is fermented in the rumen, ruminant animals depend, to a large extent, on microbial protein to meet their own protein needs. Microbial protein reaches the abomasum and small intestine when microbes are washed out of the rumen and into the lower tract. Digestive efficiency is optimized in ruminants when the growth rate of the microbial mass is maximal, resulting in maximal delivery of microbial protein to the host animal. These conditions are best met by rapidly growing populations of microbes. The microbial growth rate depends on the supply of nutrients and the rate at which microbes are washed from the rumen. Here we consider the effect of nutrient supply on microbial growth rate; factors affecting the rate of microbial removal are discussed later.

The overall reaction in the rumen may be greatly simplified, for the purposes of this discussion, to Equation 1:

glucose+peptide=microbes+VFA+NH3+CH4+CO2 (1)

(1)

Glucose and peptide represent ruminally available carbohydrate and protein, respectively. In this context, available means available to the microbes for fermentation. Carbohydrate or protein that is not susceptible, or accessible, to microbial attack is classified as unavailable and is not included in Equation 1. Glucose was chosen to represent carbohydrate, and peptide to represent protein, because all carbohydrates must be broken down to simple sugars, and proteins to peptides, before becoming available to bacteria. The term peptide in this equation could be replaced by other forms of nitrogen, but for now, the discussion is confined to peptide as a nitrogen source. Peptide is the only nitrogen-containing substrate on the left in the equation, but there are two nitrogen-containing products on the right: microbes (as protein) and NH₃ (ammonia). Both substrates, glucose and peptide, contain carbon, oxygen, and hydrogen and thus can contribute to the formation of microbial carbon, VFA, CH₄, and CO₂.

Equation 1 always balances, but the distribution of products varies according to the relative concentrations of substrates, as illustrated in Figure 31-5. For microbial cells to be produced, both energy and nitrogen are required. Energy can come from either glucose or peptide, but nitrogen must come from peptide. When glucose and peptide availability are appropriately matched (Figure 31-5, A), energy for cellular growth comes primarily from glucose, with peptides directed toward microbial protein synthesis. Under these conditions, the products of Equation 1 favor microbial cells with little ammonia production. Glucose fermentation with accompanying VFA production must be high to meet the large energy demands necessary to support the rapid growth of the microbial mass. Ammonia production is low because most peptide nitrogen is being incorporated into microbial protein.