LAB 5. Fermentation and Respiration

1 lab 5 . Fermentation and RespirationProtocols for Anaerobic growth, including use of Anaerobe Chamber, Catalase Assay, Oxidase Assay,Assay for Carbohydrate Utilization, Use of Oxidative- Fermentation that use preformed organic compounds as their source of carbon andenergy are called chemoheterotrophs. Chemoheterotrophs exhibit two basicstrategies of energy production: Respiration and Fermentation . To produce energy, bothprocesses depend upon the flow of electrons from a substrate to a terminal electronacceptor. The electron acceptor (having a negative reduction potential) accepts electronsreadily from the substrate (having a positive reduction potential).

2 Respiration is themore efficient process whereby the substrate molecule is completely oxidized to CO2,and a maximum amount of energy is released. This process most commonly uses aninorganic molecule to serve as the final electron acceptor (O2 in aerobic Respiration ). Fermentation is less efficient. The substrate, commonly a sugar such as glucose, is notcompletely oxidized. Rather, the substrate is broken down to form pyruvate which stillharbors potential energy. In Fermentation the electrons from oxidation flow to an organicelectron acceptor (pyruvate, or a byproduct of pyruvate) resulting in the formation offermentation end products.

3 The chemistry of endproducts formed during fermentationwill vary depending upon the steps in this second portion of the Fermentation diversity within heterotrophic organisms can be seen in the pathways used forenergy metabolism ( Respiration and/or Fermentation ), in the substrates used, in thespecific pathways available, and in the products produced. Particularly withfermentation, a variety of pathways have been observed among chemoheterotrophs. Asthese pathways produce characteristic products such as organic acids (lactic, acetic,butyric, propionic); neutral products (acetone, butyl alcohol, ethyl alcohol); and variousgases (carbon dioxide, hydrogen, and methane), Fermentation pathways are commonlynamed for the endproducts produced Mixed Acid Fermentation , and ButanediolFermentation.

4 Characteristics of energy producing pathways are important inclassification and identification of of ProtocolsAssay for oxidation/ Fermentation catabolism - OF media Page 83OF media is used to distinguish whether chemoheterotroph utilizes carbohydratesaerobically (Oxidation) or anaerobically ( Fermentation ). The direct oxidation pathway isan alternative to the Embden Meyerhoff pathway of glycolysis and occurs primarily instrict aerobes. To distinguish between an organism capable of Fermentation , and one thatwill perform oxidation, the OF test is performed.

5 OF media contains a small amount ofpeptone, a test carbohydrate (such as glucose) and the pH indicator bromothymol test organism is inoculated into two identical OF tubes. Tube 1 is sealed withsterile mineral oil to create an anaerobic environment. Tube 2 is exposed to the incubation, the tubes are observed for acidity. Acidity in the open tube only,indicates oxidation. Acidity in both tubes, indicates either Fermentation only, or bothfermentation and oxidation. If neither tube is acidified, the organism is unable tocatabolize the for Carbohydrate Utilization Page 69 Data from the OF test will reveal if organisms are capable of Fermentation .

6 In thesecond period of this lab you will have the opportunity to examine more closely thecharacteristics of any Fermentation positive organism. The range of sugars fermented by aparticular organism is commonly used as an assay to identify and characterize example, E. coli can be discriminated from other Gram negative fermentativeorganisms such as Serratia marcescens, and Proteus vulgaris by observing the patternof sugars each is capable of ability of organisms to ferment carbohydrates is determined by observing theproduction of acid (+/- gas).

7 This is accomplished by using a medium that contains a pHindicator and an inverted tube that can catch evolved gas. A commonly used pHindicator is phenol red. Phenol red is red at pH (the original pH of the medium), andyellow below pH for catalase enzyme Page 71 The presence of the enzyme catalase allows organisms to break down toxicproducts of oxygen. Most organisms that tolerate atmospheric oxygen will have thisenzyme. Another enzyme that destroys toxic products of oxygen is ---------> 2H2O + O2 catalaseCatalase is detected through a slide test.

8 Hydrogen peroxide is added to a small amountof culture on a glass slide. The evolution of bubbles (oxygen) indicates the presence ofthe enzyme for oxidase enzyme Page 82 Oxidase enzyme, or cytochrome oxidase, is found in the electron transport chainof many bacteria. If the test for oxidase is positive, the test organism has an electrontransport chain with cytochrome oxidase. If the test is negative, the organism may havean electron transport chain, but it will not contain cytochrome test for oxidase, an artificial substrate (p-phenylenediamine) is added to theculture.

9 If the organism has the oxidase enzyme, an electron will be passed to theartificial substrate, reducing it, and causing a change in color (from colorless to blue). Assay for Growth in presence and absence of oxygen Page 50As mentioned in Lab 4 the an oxygen requirement for growth relates to the energymetabolism of an organism. An aerobe will use Respiration with oxygen as the terminalelectron acceptor. A heterotrophic anaerobe will perform Fermentation , which does notrequire oxygen. A facultative anaerobe will have the option to ferment in the absence ofoxygen or respire in its variety of tactics are available to grow anaerobes in the laboratory:(1)Growth in media containing a strong reducing agent such as sodiumthioglycolate.

10 Sodium thioglycolate has a low oxidation reductionpotential which allows the sulfhydryl groups of this chemical to bindoxygen and allow anaerobes to grow.(2)Growth in an environment without oxygen. Such an environment can befound in:a. the bottom of agar deeps, where little air circulation occurs, orb. in an anaerobic culture jar that can be used to excludeatmospheric Gas Pak anaerobic culture system uses a jar with a sealed envelopes placed in the jar generate hydrogen. The hydrogenreacts with oxygen to produce water. The reaction requires a palladiumcatalyst.