(Solved): Carbohydrate Fermentation LEARNING OBJECTIVES Once you have completed this experiment, you should ...

Carbohydrate Fermentation LEARNING OBJECTIVES Once you have completed this experiment, you should be able to 1. Distinguish between cellular respiration and fermentation. 2. Determine how microorganisms degrade. and ferment carbohydrates by producing acid and gas. Principle Most microorganisms obtain their energy through a series of orderly and integrated enzymatic reac- tions leading to the biooxidation of a substrate, frequently a carbohydrate. The major pathways involved are shown in Figure 20.1. Organisms use carbohydrates differently depending on their enzyme complement. Some organisms are capable of fermenting sugars such as glucose anaerobically, while others use the aer- obic pathway. Still others, facultative anaerobes, are enzymatically competent to use both aerobic and anaerobic pathways, and some organisms lack the ability to oxidize glucose by either path- way. In this exercise, we'll focus on fermentative pathways. In fermentation, substrates such as carbohy- drates and alcohols undergo anaerobic dissimila- tion and produce an organic acid (for example, lactic, formic, or acetic acid) that may be accom- panied by gases such as hydrogen or carbon diox- ide. Facultative anaerobes are usually the so-called fermenters of carbohydrates. Fermentation is best described by the degradation of glucose by way of the Embden-Meyerhof pathway, also known as the glycolytic pathway, illustrated in Figure 20.2. As the diagram shows, one mole of glucose is converted into two moles of pyruvic acid, which is the major intermediate compound produced by glucose degradation. Subsequent metabolism of pyruvate is not the same for all organisms, and a variety of end products result that define their dif- ferent fermentative capabilities. This can be seen in Figure 20.3. EXPERIMENT 20 Aerobic: Biooxidations in which molecular oxygen can serve as the final electron acceptor. Cellular respiration Anaerobic: Biooxidations in which inorganic ions other than oxygen, such as NO? or SO 2, can serve as the final electron acceptors. Fermentation A biooxidative process not requiring oxygen in which an organic substrate serves as the final electron acceptor. Figure 20.1 Biooxidative pathways Fermentative degradation under anaerobic conditions is carried out in a fermentation broth tube containing a Durham tube, an inverted inner vial for the detection of gas production, as illus- trated in Figure 20.4. A typical carbohydrate fer- mentation medium contains 1. Nutrient broth ingredients for the support of the growth of all organisms 2. A specific carbohydrate that serves as the sub- strate for determining the organism's fermen- tative capabilities 3. The pH indicator phenol red, which is red at a neutral pH (7) and changes to yellow at a slightly acidic pH of 6.8, indicating that slight amounts of acid will cause a color change. Because of the critical nature of the fermen- tation reaction and the activity of the indicator, all cultures should be observed within 48 hours. Extended incubation may mask acid-producing reactions by production of alkali because of enzymatic action on substrates other than the carbohydrate. Following incubation, carbohydrates that have been fermented with the production of acidic wastes will cause the phenol red (Figure 20.5a) to turn yellow, thereby indicating a positive reaction (Figure 20.5b and Figure 20.5c). In some cases, quocino Chad Welsh. ADP ATP ADP ATP 2H?PO4 2ATP-2ADP 2ATP2ADP - 2ATP + 4ATP Net = + 2ATPs Figure 20.2 The Embden-Meyerhof pathway Organisms Lactic acid bacteria CHO Enteric bacteria Glucose Pyruvic acid 2C? Yeast (Saccharomyces) Acetic acid bacteria Respiration- Clostridia ?Amination (+NH?) -Amino acids Acetyl + SCOA Acetyl SCOA - Figure 20.3 Variations in the use of pyruvic acid acid production is accompanied by the evolution of a gas (CO?) that will be visible as a bubble in the inverted tube (Figure 20.5b). Cultures that are Fermentation Starting reactions ATP-generating reactions (C6) (C?-P) Glucose Glucose-6-phosphate Fructose-6-phosphate (C?-P) Fructose-1,6-diphosphate (P-C6-P) (2C?-P) 2 Phosphoglyceraldehyde (PGAL) 2NAD+ 2NADH? 2 Diphosphoglyceric acid ? 2 Phosphoglyceric acid 2 Pyruvic acid 2(P-C?-P) 2(C?-P) 2(C3) +2H 2CO? + 2C? Alcohol (in plants and many organisms) Products +2H 2NADH? 2NAD+ 2C3 Lactic acid (in animals and some organisms) Streptococcus Lactic and formic acids and ethanol Lactobacillus Escherichia coli Enterobacter aerogenes Salmonella typhi Ethanol, CO?, and H?; formic, lactic, acetic," and succinic acids Enterobacter aerogenes | Acetylmethylcarbinol (acetoin) S. cerevisiae | Acetaldehyde and CO? S. carlsbergensis ethanol Acetobacter Acetic acid C. beijerinckii C. acetobutylicum Butyric acid, butyl alcohol, acetone, ethanol, CO?, and H? Acetoacetic acid beta-hydroxybutyric acid Fats Krebs cycle Energy production not capable of fermenting a carbohydrate sub- strate will not change the indicator, and the tubes will appear red; there will not be a concomitant Fermentation broth Incubation ? Durham tube- Gas No gas Figure 20.4 Detection of gas production evolution of gas. Figure 20.5d illustrates this nega- tive reaction. The lack of carbohydrate fermentation by some organisms should not be construed as absence of growth. The organisms use other nutrients in the medium as energy sources. Among these nutrients are peptones present in nutrient broth. Peptones can be degraded by microbial enzymes to amino acids that are in turn enzymatically converted by oxidative deamination to ketoamino acids. These are then metabolized through the Krebs cycle for energy production. These reactions liberate ammo- nia, which accumulates in the medium, forming ammonium hydroxide (NH4OH) and producing an alkaline environment. When this occurs, the phenol red turns to a deep red in the now basic medium. Figure 20.6 illustrates this alternative pathway of aerobic respiration. CLINICAL APPLICATION Using Fermentation Products to Identify Bacteria The fermentation of carbohydrates allows microbiologists to identify some bacteria by determining what nutrients they are using and what products they produce. The pattern of sugars fermented may be unique to a particular genus, species, or strain. Lactose fermentation is one test that distinguishes between enteric and non-enteric bacteria. Dextrose fermentation allows for the differentiation between the oxidase (+) Vibrio and Pseudomonads species in patients suffering from septicemia after eating contaminated fish. (a) (b) (c) (d) Figure 20.5 Carbohydrate fermentation test. (a) Uninoculated, (b) acid and gas, (c) acid, and (d) negative. FURTHER READING Refer to the section on cellular metabolism and metabolic pathways in your textbook for further information on bacterial metabolism and growth. In your textbook's index, search under "Fermenta- tion," "Embden-Meyerhof Pathway," and "Cellular Respiration." AT THE BENCH Materials Cultures For the short version, 24- to 48-hour Trypticase soy broth cultures of Escherichia coli Alcaligenes faecalis Salmonella typhimurium BSL-2 Staphylococcus aureus BSL-2 For the long version, 24- to 48-hour Trypticase soy broth cultures of the 13 organisms. Media For the short version, per designated student group 5ml tubes (each with Durham tubes): Phenol red lactose broth Dextrose (glucose) broth Sucrose broth For the long version, 14 of each. Amino acids NH? COOH CHÁNH, CH3 (Alanine) Oxidative deamination NH? COOH CHÁNH, CH, COOH (Aspartic acid) Glucose CH?-C-COOH (Pyruvic acid) Ö CH?-C COA (Acetyl-CoA) H?-(~ COOH C=O Krebs cycle CH? COOH (Oxaloacetic acid) REACTION Proteins Figure 20.6 Proteins as energy sources for microbes Equipment Microincinerator or Bunsen burner Inoculating loop Glassware marking pencil Controls Sugar Acid Acid w/Gas Dextrose S. aureus E. coli Sucrose S. aureus K. pneumoniae E. coli Lactose S. aureus Procedure Lab One 1. Using aseptic technique, inoculate each experimental organism into its appropriately labeled medium by means of loop inoculation. Oxidative deamination •COOH C =0 CH? CH? COOH (a-ketoglutaric acid) COOH CHÁNH, CH? CH? COOH (Glutamic acid) Note: Take care during this step not to shake the fermentation tube; shaking the tube may accidentally force a bubble of air into the inverted gas vial, displacing the medium and possibly rendering a false-positive result. The last tube will serve as a control. 2. Incubate all tubes for 24 hours at 37°C. Procedure Lab Two 1. Examine all carbohydrate broth cultures for color and the presence or absence of a gas bubble. Record your results in the chart pro- vided in the Lab Report. 2. Based on your observations, determine and record whether each organism was capable of fermenting the carbohydrate substrate with the production of acid or acid and gas. Citric acid Amino acids NH? Name: Date: Section:. Observations and Results Lactose Observation (color of medium, bubble in fermentation tube) Result Bacterial Species (A), (A/G), or (-) E. coli A. faecalis S. typhimurium S. aureus K. pneumoniae P. vulgaris P. aeruginosa E. aerogenes M. luteus L. lactis S. dysenteriae B. cereus C. xerosis Alternate organism Control Dextrose Observation (color of medium, bubble in fermentation tube) Result (A), (A/G), or (-) EXPERIMENT 20 Lab Report Result (A), (A/G), or (-) Sucrose Observation (color of medium, bubble in fermentation tube)