Catabolic Reactions

Organic nutrient substrates are catabolized in a wide variety of enzymatic processes that can be schematically divided into four phases:

Digestion. Bacterial exoenzymes split up the nutrient substrates into smaller molecules outside the cell. The exoenzymes represent important pathogenic-ity factors in some cases.

Uptake. Nutrients can be taken up by means of passive diffusion or, more frequently, specifically by active transport through the membrane(s). Cytoplasmic membrane permeases play an important role in these processes.

Preparation for oxidation. Splitting off of carboxyl and amino groups, phosphorylation, etc.

Oxidation. This process is defined as the removal of electrons and H+ ions. The substance to which the H2 atoms are transferred is called the hydrogen acceptor. The two basic forms of oxidation are defined by the final hydrogen acceptor (Fig. 3.15).

■ Respiration. Here oxygen is the hydrogen acceptor. In anaerobic respiration, the O2 that serves as the hydrogen acceptor is a component of an inorganic salt.

■ Fermentation. Here an organic compound serves as the hydrogen acceptor.

The main difference between fermentation and respiration is the energy yield, which can be greater from respiration than from fermentation for a given nutrient substrate by as much as a factor of 10. Fermentation processes involving microorganisms are designated by the final product, e.g., alcoholic fermentation, butyric acid fermentation, etc.

The energy released by oxidation is stored as chemical energy in the form of a thioester (e.g., acetyl-CoA) or organic phosphates (e.g., ATP).

162 3 General Bacteriology — Bacterial Oxidation Pathways

Energy Produced From Butyric Acid

Fig. 3.15 In oxidation of organic nutrient substrates, protons (H+) and electrons (e-) are transferred in more or less long chains. The respiration is aerobic when the final electron acceptor is free oxygen. Anaerobic respiration is when the electrons are transferred to inorganically bound oxygen. Fermentation is the transfer of H+ and e- to an organic acceptor.

Anaerobic respiration

- Nitrate respiration

- Sulfate respiration

Aerobic Respiration

Fig. 3.15 In oxidation of organic nutrient substrates, protons (H+) and electrons (e-) are transferred in more or less long chains. The respiration is aerobic when the final electron acceptor is free oxygen. Anaerobic respiration is when the electrons are transferred to inorganically bound oxygen. Fermentation is the transfer of H+ and e- to an organic acceptor.

The role of oxygen. Oxygen is activated in one of three ways:

■ Transfer of 4e- to O2, resulting in two oxygen ions (2 O2-).

■ Transfer of 2e- to O2, resulting in one peroxide anion (1 O22-).

■ Transfer of 1e- to O2, resulting in one superoxide anion (1 O2-).

Hydrogen peroxide and the highly reactive superoxide anion are toxic and therefore must undergo further conversion immediately (see Fig. 3.15).

Bacteria are categorized as the following according to their O2-related behavior:

■ Facultative anaerobes. These bacteria can oxidize nutrient substrates by means of both respiration and fermentation.

■ Obligate aerobes. These bacteria can only reproduce in the presence of O2.

■ Obligate anaerobes. These bacteria die in the presence of O2. Their metabolism is adapted to a low redox potential and vital enzymes are inhibited by O2.

■ Aerotolerant anaerobes. These bacteria oxidize nutrient substrates without using elemental oxygen although, unlike obligate anaerobes, they can tolerate it.

Basic mechanisms of catabolic metabolism. The principle of the biochemical unity of life asserts that all life on earth is, in essence, the same. Thus, the catabolic intermediary metabolism of bacteria is, for the most part, equivalent to what takes place in eukaryotic cells. The reader is referred to textbooks of general microbiology for exhaustive treatment of the pathways of intermediary bacterial metabolism.

Anabolic Reactions

It is not possible to go into all of the biosynthetic feats of bacteria here. Suffice it to say that they are, on the whole, quite astounding. Some bacteria (E. coli) are capable of synthesizing all of the complex organic molecules that they are comprised of, from the simplest nutrients in a very short time. These capacities are utilized in the field of microbiological engineering. Antibiotics, amino acids, and vitamins are produced with the help of bacteria. Some bacteria are even capable of using aliphatic hydrocarbon compounds as an energy source. Such bacteria can "feed" on paraffin or even raw petroleum. It is hoped that the metabolic capabilities of these bacteria will help control the effects of oil spills in surface water. Bacteria have also been enlisted in the fight against hunger: certain bacteria and fungi are cultivated on aliphatic hydrocarbon substrates, which supply carbon and energy, then harvested and processed into a protein powder (single cell protein). Culturing of bacteria in nutrient mediums based on methanol is another approach being used to produce biomass.

Metabolic Regulation

Bacteria are highly efficient metabolic regulators, coordinating each individual reaction with other cell activities and with the available nutrients as economically and rationally as possible. One form such control activity takes is regulation of the activities of existing enzymes. Many enzymes are allosteric proteins that can be inhibited or activated by the final products of metabolic pathways. One highly economical type of regulation controls the synthesis of enzymes at the genetic transcription or translation level (see the section on the molecular basis of bacterial genetics (p. 169ff.).

Growth and Culturing of Bacteria

Nutrients

The term bacterial culture refers to proliferation of bacteria with a suitable nutrient substrate. A nutrient medium (Table 3.2) in which chemoorgano-trophs are to be cultivated must have organic energy sources (H2 donors) and H2 acceptors. Other necessities include sources of carbon and nitrogen for synthesis of specific bacterial compounds as well as minerals such as sulfur, phosphorus, calcium, magnesium, and trace elements as enzyme activators. Some bacteria also require "growth factors," i.e., organic compounds they are unable to synthesize themselves. Depending on the bacterial species involved, the nutrient medium must contain certain amounts of O2 and CO2 and have certain pH and osmotic pressure levels.

Table 3.2 Nutrient Mediums for Culturing Bacteria Nutrient medium Description

Nutrient broth Complex liquid nutrient medium.

Nutrient agar Complex nutrient medium containing the polysaccharide agarose (1.5-2%). Nutrient agar liquefies when heated to 100 °C and does not return to the gel state until cooled to 45 °C. Agarose is not broken down by bacteria.

Selective mediums Contain inhibitor substances that allow only certain bacteria to proliferate.

Indicator mediums Indicate certain metabolic processes. Synthetic mediums Mediums that are precisely chemically defined.

Growth and Cell Death

Bacteria reproduce asexually by means of simple transverse binary fission. Their numbers (n) increase logarithmically (n = 2G). The time required for a reproduction cycle (G) is called the generation time (g) and can vary greatly from species to species. Fast-growing bacteria cultivated in vitro have a generation time of 15-30 minutes. The same bacteria may take hours to reproduce in vivo. Obligate anaerobes grow much more slowly than aerobes; this is true in vitro as well. Tuberculosis bacteria have an in-vitro generation time of 12-24 hours. Of course the generation time also depends on the nutrient content of the medium.

The so-called normal growth curve for bacteria is obtained by inoculating a nutrient broth with bacteria the metabolism of which is initially quiescent, counting them at intervals and entering the results in a semilog coordinate system (Fig. 3.16). The lag phase (A) is characterized by an increase in bacterial mass per unit of volume, but no increase in cell count. During this phase, the metabolism of the bacteria adapts to the conditions of the nutrient medium. In the following log (or exponential) phase (C), the cell count increases logarithmically up to about 109/ml. This is followed by growth deceleration and transition to the stationary phase (E) due to exhaustion of the nutrients and the increasing concentration of toxic metabolites. Finally, death phase (F) processes begin. The generation time can only be determined during phase C, either graphically or by determining the cell count (n) at two different times and applying the formula:

— Normal Growth Curve of a Bacterial Culture log2 n2 - log2 ni '

— Normal Growth Curve of a Bacterial Culture

Deceleration Phase Growth Curve

Fig. 3.16 A = lag phase, B = acceleration phase, C = log (exponential) phase, D = deceleration phase, E = stationary phase, F = death phase.

(Hours)

Time

(Hours)

Time

Fig. 3.16 A = lag phase, B = acceleration phase, C = log (exponential) phase, D = deceleration phase, E = stationary phase, F = death phase.

Bacterial Cell Count and Bacterial Mass

The colony counting method. The number of living cells in a given culture or material can be determined by means of the colony counting method. The samples are diluted logarithmically by a dilution factor of 10. Using the pour plate technique, each dilution is mixed with 1 ml of liquid agar and poured out in a plate. In the surface inoculation method, 0.1 ml of each dilution is plated out on a nutrient agar surface. The plates are incubated, resulting in colony growth. The number of colonies counted, multiplied by the dilution factor, results in the original number of viable bacterial cells (CFU = colony forming units).

Bacterial mass. The bacterial mass can be established by weighing (dry or wet weight). The simplest way to determine the mass is by means of photometric adsorption measurement. The increases in mass and cell count run parallel during phase C on the growth curve.

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Responses

  • alina
    WHAT ARE THE CATABOLIC REACTIONS THAT CELLS USE TO GET ENERGY FROM NUTRIENTS?
    7 years ago
  • mika
    Which of the following is not a catabolic reaction that cells use to get energy from nutrients?
    7 years ago

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