Case Study I—Soy Sauce. Scenario: Brewing soy sauce is one of the original biotech industries. Soy sauce was shipped in barrels within Asia over 500 years ago, and in bottles to Europe by the 1600s. Now soy sauce is used all over the world. About 5000 years ago in China, people grew soybean crops for food and animal feed. Because soybeans spoil easily, salt was added as a preservative. Over time the beans fermented much like pickles or sauerkraut. Unlike pickles, however the soy beans turn into a paste called miso as they ferment. The paste is easier to digest than the unfermented soy beans, and people have been eating it for centuries. About 500 years ago, someone discovered that instead of discarding the sauce at the bottom of the barrels, theycould use it for cooking. Thus, soy sauce was invented. Unlike making wine from grapes, soy sauce brewing is performed in two stages. First, the soy beans are steamed and mixed with toasted crushed wheat. Fungi Aspergillus oryzae and Aspergillus sojae are added to the mixture to make koji (the first step in the soy sauce-making process) that is then left uncovered for a couple of days. Next, salt and water are added to koji to form a mash called moromi. Moromi is then put in airtight containers where it is allowed to ferment for at least 6 months. The mash is then squeezed to get the liquid soy sauce. Finally, the sauce is filtered, pasteurized, and tightly bottled for distribution. Questions I. Koji phase 1. In the koji phase, the mixture is inoculated with two species of Aspergillus fungi, and the mixture is left uncovered. i) If you analyze the microbial populations found in koji, what do you expect to find? A. Aspergillus fungi only B. Aspergillus and other fungi C. bacteria only D. various fungi (including Aspergillus) and bacteria E. no microbes ii) If you expect to find microbes other than the ones with which the mixture was inoculated, explain where they come from and why they thrive in koji. If you do not expect to find additional populations, explain why not. Because the koji mixture is left uncovered, microbes other than what the mixture was inoculated with have a chance to fall into the vats. And because many nutrients are available in this complex mixture, these various species can thrive in koji.2. In the koji phase of soy sauce production, the content of the bean/wheat paste and the number of fungi change. These two processes are A. related to each other in that the rate of one is dependent on the rate of the other B. related to each other in that both are caused by the same third process C. unrelated to each other 3. What reactions/processes (if any) are responsible for changes described in question 2 above? Macromolecules found in koji are broken down via hydrolysis reactions, and some of the glucose generated is utilized through glycolysis and respiration by the various organisms in the mix to produce ATP for metabolism and reproduction. 4. Where in the cell do these reactions/processes occur? The hydrolysis reaction that break down macromolecules actually happen outside of the cells. Enzymes secreted by the two Aspergillus species are particularly important in catalyzing the breakdown of macromolecules. Glycolysis proceeds in the cytoplasm of individual cells, citric acid cycle and ETC occur in the mitochondria. II. Moromi phase Once Aspergillus has broken down the macromolecules in the soybeans and wheat into monomers, the koji phase ends. Moromi is then made by mixing koji with water and enough salt to make a 16-20% concentrated salt solution, or brine. 1. When brine is added, the populations of microbes found in koji change. Do you expect greater or lesser microbial diversity? A. Greater B. Lesser 2. How does adding brine lead to these changes? Most microbial species can’t survive at high salt concentrations because osmosis causes water from inside the cell (low salt environment) to leak out into the high salt environment of the outside. 3. Another challenge to the microbes in moromi is that it is placed in airtight containers for a number of months. What types of microbes will survive under these conditions? Organisms that are either always anaerobic in their metabolism or those that can switch to anaerobic metabolism. Explain how they obtain energy for life processes. Glycolysis does not require oxygen, so organisms use glycolysis to derive their ATP. But because this type of metabolism requires that NADH molecules are recycled back to NAD+, pyruvate is fermented to either lactic acid or ethanol in the reaction that recycles NADH back to NAD+.4. Both lactic acid and ethanol are found in soy sauce after the moromi phase is complete. At a minimum, how many species of microbes thrive in moromi? A. 0 B. 1 C. 2 D. 3 E. can’t tell Justify your answer. Fermentation pathways end up with either lactic acid or ethanol as waste products. Because both are present in moromi, we know that both types of fermentation have to be performed by organisms in the mixture, i.e. there are at least two types of microbes at work here. 5. Lactic acid and ethanol are produced from the same starting material. i) What is the starting molecule? A. Glucose B. Pyruvate C. ATP D. NADH E. CO2 F. Other ii) What other waste products are produced along with lactic acid and/or ethanol? In the ethanol fermentation, pyruvate is first converted to a substance that can be converted to ethanol, releasing CO2 as a waste product along the way. iii) Do lactic acid and ethanol result from oxidation or reduction of their respective precursors? A. Lactic acid and ethanol both result from oxidation of the precursors B. Lactic acid and ethanol both result from reduction of the precursors C. Lactic acid results from oxidation, while ethanol results from reduction D. Lactic acid results from reduction, while ethanol results from oxidation III. Alcohol concentrations Fermentation easily occurs without human intervention. For example, tiny breaks on the skin of ripened grapes allow entry of microbes. Fermentative yeasts thrive in the interior of the grape, which provides both the high concentration of sugars and low pH. Yeasts metabolize the grape sugars for energy, and the waste products—carbon dioxide and ethanol—are rapidly transported out of the cell. In an artificial fermentation, such as making wine, the process is carried out in a manner that permits CO2 to
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