Silos and Sauerkraut

Transcription

1 LessonS ~ -----\_rj Silos and Sauerkraut Investigating microbial fermentation Part I: Factors in fermentation Part II: Mixing in some microbes Overview Throughout history, humans have harnessed fermentation to produce and preserve food. In biblical times, people fermented grapes into wine, and used fermentation to store grain. Today we rely on microbial fermentation to bring us yogurt, yeast breads, cheese, sauerkraut, kimchee, wine and beer. Microbial fermentation is also important in agriculture. In order to preserve and store high-quality feed for livestock, farmers ferment corn plants to produce silage, and hay to produce haylage. In this way, succulent forage crops are preserved and stored for use throughout the year. This activity is divided into two parts. Part I, Factors in fermentation, examines the environmental variables critical to the fermentation process. In Part II, Mixing in some microbes, students investigate the role of microorganisms in fermentation by manipulating the microbial populations and observing fermentation results. Students build minisilos out of soda bottles and ferment their own products while carefully monitoring the "silo's" temperature, ph, and gas production. Biological and agricultural concepts Microbiology The carbon cycle The nitrogen cycle Decomposition Symbiosis Anaerobic fermentation ph Food production Scientific method Fermentation I 5-1

2 Teacher material Science teachers, both in agriculture and biology, can use this ex- periment to introduce the topics of microbiology and biochemistry as they apply to food production. The teachable moment Many biology teachers begin their biology program with a unit on cell biology and cell chemistry. Fermentation experiments can illustrate cell respiration and microbial activity. These experiments can extend further into topics such as food or feed processing, preservation, and animal feed augmentation. You might also use this activity to lead into discussions of new technologies that use microbial genetic engineering. You might consider using this laboratory in the first few weeks of class as an introduction to scientific method. Students will be challenged to develop critical thinking skills that will be used in determining the relationship between bacteria and fermentation. Background Fermentation or the "pickling" of food as a method of preservation has been around since ancient times. Fermentation, if carried out correctly, preserves valuable nutrients in food and allows one to store food with minimal loss in quality. Fermentation is the anaerobic process by which bacteria convert complex compounds, such as sugars, into simpler products, such as alcohol, lactic acid and carbon dioxide. While these products of fermentation are often quite palatable to humans and other animals, their presence inhibits the growth of other non-fermentative bacteria. Fermentation thus prevents food from spoiling by inhibiting the growth of undesirable bacteria and fungi. Many different bacteria can play a part in fermentation. A common group of bacteria, called Lactobacillus, convert sugars, found in plant cell walls, to lactic acid, alcohol, carbon dioxide and other simple sugars. Some lactic acid bacteria, or the homofermentative variety, convert most sugars directly into basically a single product, lactic acid. Other bacteria, those that are heterofermentative, convert the sugars into lactic acid, alcohol, carbon dioxide, and other sugars. When populations of the homofermentative bacteria grow, there is a rapid decrease in ph (due to the acid production), but little C0 2 gas is produced and little temperature change takes place. Alternatively, when populations of the heterofermentative bacteria are growing, you will see a slower decrease in ph, followed by a subse- 5-2 I Fermentation

3 Teacher material quent rise in ph, a greater change in temperature and an increased level of C0 2 production. Thus, depending on the desired fermentation product, homofermentors may be more desirable, due to the fact that less heat and gas are produced and there is a rapid decrease in ph. The process of fermentation is not limited to the production of human food. The agricultural industry uses this valuable process to ferment plant material to produce feed, or forages, for animals. Forages are those feeds high in fiber and low in digestibility, such as hay or grass. Forages are an excellent source of nutrients, can often be grown on land unsuitable for human crop production and are much cheaper per acre than grain. Because preserving high-quality forages for use in winter can help reduce the costs associated with feeding and raising animals, a great deal of emphasis is now being placed on production of high quality forage crops. The fermentation of crops takes place when they are ensiled, or placed into a silo. The word silo is derived from the Greek word meaning "a pit or hole sunk in the ground for storing corn." The production of a high-quality silage product requires that plant respiration, proteolytic (protein degrading) enzyme activity, clostridial activity (undesirable anaerobic bacterial growth) and aerobic microbial growth be limited. An essential way to limit these processes is to create anaerobic (lacking oxygen) conditions within the silo. Harvested plant material in the presence of oxygen means that aerobic microbial activity can take place and that the material, if left exposed, will decay to a useless, inedible and often toxic product. Filling a silo rapidly, making sure the plant material is very compact, and tightly sealing the silo keeps the oxygen level to a minimum. The amount of moisture in the silage is another important factor in achieving a high quality fermentation product. For crops ensiled with greater than 45 percent moisture, a rapid decline in ph is essential to control growth of the bacteria Clostridia. Clostridia sp. produces butyric acid as a metabolic waste product and degrades protein to a variety of products that have poor nutritional value. Because temperatures in the range of F will also encourage the growth of Clostridia, temperatures higher than 99 degrees F should be maintained, if possible. Corn silage is normally ensiled at a moisture content of percent. Grasses and legumes are ensiled at percent moisture. With time, oxygen-consuming microorganisms in a packed silo create an increasing anaerobic environment in the silo. Under these condi- Fermentation I 5-3

4 Teacher material tions, the beneficial bacteria present on the crops, Lactobacilli, begin to multiply. Although Lactobacilli can use oxygen, they are considered facultative anaerobes and are able to survive and reproduce in anaerobic conditions. Clostridia, however, require strict anaerobic conditions and cannot tolerate a low ph environment. So in the beginning of the fermentation process in a silo, the Lactobacilli are able to grow in a slightly aerobic environment. As the Lactobacilli convert sugars to lactic acid, acetic acid and ethanol, the ph drops. As the silo becomes anaerobic, the Lactobacilli have had a head start on the Clostridia, and have begun to lower the ph, making it hard for the Clostridia to grow and compete. In a ph range of 3.7 to 4.1, silage will be preserved almost indefinitely. The fermentation process usually takes one to three weeks. While lactic acid bacteria are found on foods and plants naturally, they do not always exist in high enough numbers to ensure that the fermentation process will be successful. By adding favorable bacteria, often termed additives or inoculants, you can be more confident that there will be enough desirable strains of bacteria present to outcompete undesirable strains. The use of additives often produces a higherquality fermentation, thus producing a superior stored product. Although research does not show that using an inoculant produces any increase in the nutritional value of feed, treated silage is more palatable and has a longer bunk, or shelf, life. In Part II of this activity, students will add yogurt, containing active cultures of Lactobacilli and commercial silage inoculants, as an additive to their silos. Continued research in ways to improve this fermentation process includes the isolation and engineering of new mutant forms of superhomofermentative bacteria to be used as silage inoculants. Also, scientists look for strains of bacteria that may work particularly well on particular crops I Fermentation

5 Teacher material Part I: Factors in fermentation In this portion, students will explore how oxygen affects the fermentation process. They will build their own fermentation chamber, fill the chamber with plant material and monitor fermentation by measuring the ph and temperature inside the chamber. If the product is fermenting it should give off C0 2 The presence of carbon dioxide can be detected in several different ways. The first method relies on the fact that when C0 2 is bubbled through a solution, the ph of the solution decreases. Thus, the gas from a fermentation can be bubbled through water and the ph of the water can be monitored using a ph indicator. Cabbage juice works well for this! Cabbage juice, which is purple, turns red if it becomes acidic and green if it becomes basic. See Appendix C for preparation of the cabbage juice indicator solution. The second method uses limewater. When carbon dioxide is bubbling through limewater, a calcium carbonate precipitate forms. Limewater forms the white precipitate when acidified. The last method you might try is to measure the volume of the gas coming out of the fermentor by constructing a gas collector for your chamber. Instructions for this method are outlined in the procedure. Teacher Management Preparation It will take approximately two hours to collect the materials and prepare the necessary solutions. You might consider getting a student to help you. Collect the plant material12 to 24 hours prior to the investigation, to allow time for the material to dry down. Preparing solutions -If you plan to monitor the ph via C0 2 production, see appendix C, page 14, for instructions on preparing the cabbage indicator solution. Activity time Constructing the bottle chambers - It will take approximately one class period for you to demonstrate the laboratory procedure and for students to construct the bottle chambers. We suggest allowing 24 hours for the silicone on the chambers to dry completely. Chopping up the plant materials, sealing the bottle and adding the indicator solution will take one additional period. Fermentation I 5-5

6 Part 1: Teacher material Tips and safety If possible, start the materials fermenting on a Monday so that students can observe the chambers for the remainder of the week-this should take 5 to 10 minutes a day. Have the students make two or three observations the next week and one the following week. You may want to use part of an additional period for discussion of the product and discuss the results and further extensions to this laboratory activity. Remind students to carefully seal their mini-silos where the plastic tubing enters the bottle top and the upper and lower plastic bottle halves meet. Try to obtain fresh ears of com as this ensures an appropriate moisture level for the fermentation. Ideally, use whole corn plants. Chop as much of the plant as possible. If chopping the cob is too difficult, you may want to discard it. Instruct students to work slowly and carefully as they use knives to cut the corn into smalll/2-inch pieces. Push the tubing into the test tube as far as it will go so it stays in place as the gas collects. To measure the ph and temperature, students must firmly attach the ph paper to the end of the thermometer, and stick it into the hole made in the center of the chamber. Students should do this as quickly as possible to minimize the amount of oxygen entering the chamber. If you have enough thermometers, you can have students permanently place a thermometer in the silo and seal around it with silicon. Use the following guidelines to help students evaluate the quality of the final com silage product- 1. High quality silage should smell sweet and acidic, yet not too vinegary. 2. The product should have a greenish-brown color. 3. Mold should not grow in a high quality product. Black, dark brown or white areas in a product indicate mold growth. 4. The product should contain 65 to 72 percent moisture. The less loss of dry matter during the fermentation, the better your product. 5-6 I Fermentation

7 Part 1: Teacher material Materials Each group of two students will need: two clear 1-liter plastic bottles with plastic tops two small test tubes two 45 em long pieces of plastic tubing (Tygon, 5/16" O.D., 3/16" I.D.) five ears of corn (with husks) or one grocery bag of alfalfa, grass clippings or other green material tube of silicone sealant safety razor sturdy, large kitchen knife cutting board (flat piece of wood or tray is fine) holepunch sharp needle heat source to heat a hole poke balance tapered reamer or drill scissors 250 ml or larger beaker thermometer black electrical tape ph paper (to measure from a ph of 3 to 7 in increments of 0.5 units or less) lime water (calcium hydroxide) (optional) cabbage juice ph indicator solution (optional) To make the gas collector: three 60 ml syringes 10 ml syringe one 2-liter bottle paper clips (large) or tubing clamps Key terms Aerobic: containing oxygen Anaerobic: lacking oxygen Anaerobic fermentation: conversion of sugars, in the absence of oxygen, to alcohol, lactic acid or similar compounds Biochemistry: study of the chemistry of living organisms Fermentation 1 5-7

8 Part 1: Teacher material Biotechnology: integrated use of biochemistry, microbiology, and chemical engineering in order to achieve the technological application of microbes and cultured tissue cells Bunk life: the time ensiled feed remains free of mold and spoilage after it has been exposed to air Clostridia: a type of anaerobic bacteria Dry matter (DM): the material left in a food specimen after all water is removed. DM is the basis from which all feed nutrients are derived Facultative anaerobes: organisms able to grow both in the presence and absence of oxygen Lactobacillus: a type of bacteria that ferments sugars to lactic acid ph: measure of hydronium ion concentration; measure of acidity or alkalinity Silage: fodder (as of field corn, sorghum, grass, or alfalfa) either green or mature converted into succulent winter feed for livestock through processes of fermentation, usually by being cut fine and blown into an airtight chamber (silo) where it is compressed to exclude air and where it undergoes an acid fermentation that retards spoilage. Also known as ensilage References McDonald, Peter. The Biochemistry of Silage. John Wiley & Sons, Ltd.: Chichester, England Muck, R.E.. "Factors Influencing Silage Quality And Their Implications For Management," Journal of Dairy Science (ll) Pioneer Forage Manual- A Nutritional Guide. Pioneer Hi- Bred International, Inc. Des Moines, Iowa Pitt, R.E., Y. Liu and R.E. Muck. "Simulation of the Effect of Additives on Aerobic Stability of Alfalfa and Corn Silages." Transactions of the ASAE (4) Fermentation

9 Part 1: Student material Introduction Have you ever eaten sauerkraut, yogurt, pickles, sourdough bread or kimchee? Though these foods look and taste different, they have one thing in common-they are all produced through the process of microbial fermentation. This process, used to preserve the quality of food, is nothing new. Fermentation or the "pickling" of food has been around since ancient times and, if carried out correctly, preserves valuable nutrients so they can be saved and eaten in the off season or when fresh foods are not available. Fermentation is the anaerobic process by which bacteria convert complex compounds, such as sugars, into simpler products, such as alcohol, lactic acid and carbon dioxide. While these products of fermentation are often quite palatable to humans and other animals, their presence inhibits the growth of other non-fermentative bacteria. Fermentation thus prevents food from spoiling by inhibiting the growth of undesirable bacteria and fungi. The process of fermentation is not limited to the production of human food. The agricultural industry uses this valuable process to ferment plant material to produce feed, or forages, for animals. Forages are those feeds high in fiber and low in digestibility, such as hay or grass. Forages are an excellent source of nutrients, can often be grown on land unsuitable for human crop production and are much cheaper per acre than grain. Because preserving high quality forages for use in winter can help reduce the costs associated with feeding and raising animals, a great deal of emphasis is now being placed on production of high-quality forage crops. The fermentation of crops takes place when they are ensiled, or placed into a silo. The word silo is derived from the Greek word meaning "a pit or hole sunk in the ground for storing corn." The production of a high-quality silage product requires that plant respiration, proteolytic (protein degrading} enzyme activity, clostridial activity (undesirable anaerobic bacterial growth} and aerobic microbial growth be limited. An essential way to limit these processes is to create anaerobic (lacking oxygen) conditions within the silo. Harvested plant material in the presence of oxygen means that aerobic microbial activity can take place and that the material, if left exposed, will decay to a useless, inedible and often toxic product. Filling a silo rapidly, making sure the plant material is compact, and tightly sealing the silo keeps the oxygen to a minimum in the silo. Fermentation I 5-9

10 Part 1: Student material The amount of moisture in the silage is another important factor in achieving a high-quality fermentation product. For crops ensiled with greater than 45 percent moisture, a rapid decline in ph is essential to control growth of the bacteria Clostridia. Clostridia sp. produces butyric acid as a metabolic waste product and degrades protein to a variety of products that have poor nutritional value. Temperatures in the range of F will also encourage the growth of Clostridia and should be avoided. Corn silage is normally ensiled at a moisture content of percent. Grasses and legumes are ensiled at percent moisture. With time, oxygen-consuming microorganisms in a packed silo create an increasing anaerobic environment in the silo. Under these conditions, the beneficial bacteria present on the crops, Lactobacilli, begin to multiply. Although Lactobacilli can use oxygen, they are considered facultative anaerobes and are able to survive and reproduce in anaerobic conditions. Although Clostridia require strict anaerobic conditions, they cannot tolerate a low ph environment. So in the beginning of the fermentation process in a silo, the Lactobacilli are able to grow in a slightly aerobic environment. As the Lactobacilli convert sugars to lactic acid, acetic acid and ethanol, the ph drops. As the silo becomes anaerobic, the Lactobacilli have had a head start on the Clostridia, and have begun to lower the ph, making it hard for the Clostridia to grow and compete. In a ph range of 3.7 to 4.1, silage will be preserved almost indefinitely. The fermentation process usually takes one to three weeks. Materials two clear 1-liter plastic soda bottles with caps two small test tubes two 45 em lengths of plastic tubing (Tygon,5/16"0.D.,3/16"1.D.) five ears of corn (with husk) or one grocery bag of alfalfa, grass clippings or other green material tube of silicone sealant (you can also use caulking, which may be less expensive) safety razor large kitchen knife cutting board (flat piece of wood or tray is fine) hole punch sharp needle heat source for hole poke balance tapered reamer or drill (continued) Fermentation

11 Part 1: Student material scissors beaker (250 ml or larger) thermometer (type with a reading gauge on the end is best) black electrical tape ph paper (to measure from a ph of 3 to 7 in increments of 0.5 units or less) limewater (calcium hydroxide) (optional) cabbage ph indicator solution (optional) For the gas collector: three 60 ml syringes one 10 ml syringe one 2-liter bottle large paper clips or small clamps Procedure Constructing the fermentation chamber 1. Remove label and cut plastic bottle 1 em below shoulder. 2. Cut off plastic outer covering at bottom of bottle leaving a slightly visible rim on the base. This will make it easier to see into the bottom of the mini-silos. 3. Drill or ream a hole slightly smaller than the diameter of the plastic tubing in the bottle cap to allow for a tight fit of the flexible plastic tubing. 4. Punch a very small hole halfway up from the bottle base. This hole will be for temperature and ph reading. Cover hole with black electrical tape. 5. Insert plastic tubing about 2.5 em through the top of the cap. 6. Seal with silicone sealant where the plastic tubing goes through the top of the cap and let dry. To test for C Attach a test tube to the outside of the plastic bottle with silicone sealant. Fermentation I 5-11

12 Part 1: Student material 2. Fill the test tube to within 2.5 em of the top with cabbage indicator solution or limewater. 3. Place plastic tubing all the way to the bottom of the test tube. '-"''? t'ia.nc.h A. HOL.G. Jt-.1.._ 'BtJtrl..~ (j.c.66 -e'nt>\(.f.\4 Foe. 12..\.I.'B'Boe-g_ "Ike I ~6 C.IA.T 1 c.- -ae...,.~, -r- ~ tka\ol\.l>et!. ""R...t.lc.M oe?""-~"' s ~,.~ ""'"'-'" -rht5 ~--z..~ 'D "~. 'Ail(.. 1).. -n+c 'TI-l «M.ol'l ET It C."'-T ~ C.OI..oUO ~ "'Jl) 6 )C:i' D~ '6' 1'\H:' 'BO"JTl) M. 1>1" "11K: ~I' 'BI)'TI'\S ~ 5MAL.L. Tesr l\.\.."86 'f\l.u:e> WITH 1"-l'CIC.~~ SO~TlOt I Fermentation

13 Part 1: Student material Setting up the experiment 1. Cut your corn, alfalfa, grass clippings or other green plant material into 1 em pieces and then divide into two portions. You will need enough to pack your two silos tightly (approximately 2 x 500 grams) with 150 grams left over. If you are using corn, include the kernels, husk and stalks (if available) only; do not attempt to cut up the cob. 2. With the 100 grams of extra material, you will need to determine the dry matter (DM) content and initial ph of your sample. DM content is the most important outcome in silage production. A loss of DM indicates a loss of food value. If you are not going to measure DM immediately, store the sample in a sealed plastic bag to minimize moisture loss. To measure DM, microwave the sample for two minutes, stirring if necessary to prevent edges from burning. Weigh the sample and record the weight. Microwave for another 30 seconds and weigh again. Continue until the weight is constant. Calculate DM as follows: DM% = ending weight (grams) x 100 beginning weight(grams) To measure initial ph, grind 50 mls of the material, with a mortar and pestle (if available), and take the ph of the extracted liquid. 3. Firmly pack material into your bottle silos by pushing down with the knuckles of your hand as you rotate the bottle. Do this until there is 500 grams of material in each of two bottles. It is very important to pack the material as tightly as possible to eliminate all air pockets. 4. Place the tops of the bottles into the bottoms and push downward as far as they will go. 5. Seal off where the top and bottom plastic meets with silicone sealer while holding firmly in place. 6. Place the bottle caps with attached rubber tubing on top of the bottles and screw them on tightly. Fermentation I 5-13

14 Part 1: Student material 7. Using a sharp needle, poke holes in one of the bottle bases to allow for an aerobic (with oxygen) environment. The other bottle will remain anaerobic (without oxygen). 8. You will record your data every day for the first week, and on Monday, Wednesday, and Friday of the following week. One data point will be taken in the third week. You will measure and record the following: ph of the material-open the sampling port on the side of the bottle and insert ph paper. You may need to rub the paper around a bit to get moisture on the paper. Remove the paper and record the ph. Temperature of the material-insert a thermometer into the center of the silo through the sampling hole and record the temperature. Remove thermometer and tightly reseal the hole with a new piece of electrical tape. Presence of C0 2 -You can measure the presence of C0 2 by bubbling the C0 2 through water, cabbage juice or limewater. 9. Mter three weeks, measure the DM content of the anaerobic silo only. The silo exposed to oxygen may have undesirable fungi and bacteria growing in it, and should therefore not be opened and should be thrown out Fermentation

15 Part 1: Data sheets Factors in Fermentation Student name: Plant material used in silo Dry matter content % Initial ph of material: ph Temperature Day Silo 1 Silo2 Silo 1 Silo2 without oxygen with oxygen without oxygen with oxygen Comments or observations: Fermentation I 5-15

16 Part I: Data sheets Graph 1: Daily ph changes in mini-silos ph Day Graph 2: Daily temperature changes in mini-silos ph Day Discussion 1. State your hypothesis. 2. What was the variable in your experiment? 3. What happened to the ph of the control and variable during the experiment? 4. Explain your ph observations Fermentation

17 Part 1: Student material 5. What happened to the temperature of the control and variable during the experiment? 6. Explain your temperature observations. 7. Did you notice any "happenings" in your silos over the duration of the experiment? If so, what changes did you observe and what may have caused them? 8. Was your hypothesis supported? Explain. 9. What are the reactants of anaerobic fermentation? 10. What are the products of anaerobic fermentation? 11. What organisms caused the changes occurring in the mini- silos? 12. Would you describe your mini-silo system as alive and dynamic? Explain. 13. What other processes useful to people use anaerobic fermentation? Fermentation I 5-17

18 Part 1: Student material 14. Discuss your experiment with the other groups in your class that chose the same variable, compare observations, and make some summary statements based upon your combined results. Extensions 1. What would be the effect on fermentation of other variables such as feed type, system temperature, compaction, or moisture? 2. What bacteria are actually present in the final product? Try plating them out on an agar medium. 3. Are there other ph indicator solutions that could be used? 4. What other variables could be tested for? 5. Is your product palatable? Try presenting it to any ruminants on your farm. 6. What other kinds of foods/ feeds could we preserve? 7. How could a genetically-engineered form of bacteria be more efficient? 8. What applications could this have for long-term food preservation or space exploration? 9. Could you design a way to measure the type of acid produced or the type of bacteria produced? 5-18 I Fermentation

19 Part II: Teacher material Part II: Mixing in microbes In this experiment, students will observe the effects of adding a microbial inoculant to a fermentation chamber. Using 1-liter soda bottles, students build three fermentation chambers with which they will look at different inoculant additions. They will follow the fermentation process by measuring changes in temperature, ph and gas production, and observing the physical changes in the sample. Teacher management See Part I, page 5-5. Materials See materials list for Part I, page 5-7, and include the following: yogurt with active cultures commercial silage inoculant three 60 ml syringes one 10 ml syringe one 2-liter bottle large paper clips or small clamps Sources of materials A local veterinarian could be a source of syringes. Silage inoculants are available from farm supply co-operatives or a local farmer. Very small amounts are needed. Tips and safety Room temperature should be adequate for fermentation to occur. Optimum temperature is 25 to 30 C. Syringes should all be at the same level in the collector and completely covered with water to detect any leaks. Since most gas production occurs during the first four to seven days, it would work best to begin at the beginning of the week to allow five consecutive days of measurement. Fermentation I 5-19

20 Part II: Teacher material Key terms As in part I, page 5-8, and including the following: Silage inoculant: a preparation of bacterial populations that enhance the fermentation process, thus improving food quality References Henderson, A.R. "Silage making: Biotechnology on the farm." Outlook on Agriculture vol. 16 (2): Moon, N.J. A short review of the role of lactobacilli in silage fermentation. Food-Microbiology (4): "Silage inoculants and additives: great potential, variable results." Dairy Herd Management. (2):24, Silley, P. "The effect of three commercial silage additives on numbers of Lactobacilli entering the silo at the onset of fermentation." Federal European Microbiology Society.. 30 (1/2): Wood, Brian. Microbiology of fermented foods. Elsevier Science Pub. Co I Fermentation

21 Part II: Student material Introduction See Introduction to Part I on pages 5-10 and include the following: This activity illustrates anaerobic fermentation on a small scale and investigates how introducing a microbial inoculant can affect the process. Using 1-liter soda bottles, you will build three fermentation chambers. Two of the chambers will be inoculated with bacteria and the third left as a control. You will then follow the fermentation process in each chamber by measuring the changes in temperature, ph and gas production, and observing the physical changes in the sample. While lactic acid bacteria are found naturally on most foods and crops, too few of these favorable fermentative bacteria will result in a poor fermentation. The use of additives, or inoculants, can potentially enhance the quantity and quality of the fermentative bacteria, resulting in improved fermentation. Although research does not show that using an inoculant produces feed that is of higher nutritional value, treated silage is more palatable and has a longer bunk life (or shelf life). In this experiment, yogurt, containing active cultures of Lactobacilli, and a commercial silage inoculant will be added to your mini-silos. Materials five ears of corn (with husks) or one grocery bag of alfalfa, grass clippings or other green material three clear one-liter plastic bottles with plastic tops three small test tubes two 45 em lengths of plastic tubing one 6" strip of Velcro adhesive tape 20 ml of red cabbage indicator solution (or limewater) tube of silicone sealant safety razor large kitchen knife cutting board clear film can w /lid (Fuji) hole punch sharp needle heat source for hot poke balance tapered reamer or drill scissors beaker thermometer (continued) Fermentation I 5-21

22 Part II: Student material black electrical tape phpaper limewater (calcium hydroxide) (optional) cabbage ph indicator solution (optional) yogurt with active cultures commercial silage inoculant (for example, Pioneer brand 1177 or 2010, available from Microbial Genetics, Pioneer Hi-Bred International, 4601 Westown Pkwy, Suite 120, Des Moines, IA 50265) For the gas collector: three 60 ml syringes one 10 ml syringe one 2-liter bottle large paper clips or small clamps Procedure See page 5-12, steps 1 through 6, for construction of fermentation chambers, except that you will need to use longer lengths of tubinguse 18 inches of tubing coming out of each chamber. Also, do not poke holes in the fermentation chambers. Constructing the vessel for measuring amount of C0 2 produced 1. Obtain one 60 ml syringe, for each chamber you have. For each syringe, cut a short length of tubing (approx. 10 em) and insert the needle end of the syringe securely into one end of the tubing and seal with silicone. Let dry. 2. Make a hole slightly smaller than the diameter of the tubing in the side of the syringes. Insert one end of long tubing into the hole and seal with silicone. Attach the syringe to the inside of a cut bottle or beaker with silicone. (See illustration for clarity.) 3. Attach each of the syringes at the same level in the collector vessel. Fill collector vessel so the syringes fill with water. 4. Then bend the short pieces of tubing on the top of each syringe and secure with a large paper clip or clamp. 5. When you have finished constructing and filling your chambers, you will u:;e the C0 2 collection vessel by inserting the long tubing coming from each chamber up into one of the syringes. Insert the tubing all the way up into the syringe. (See illustration.) 5-22 I Fermentation

23 Part II: Student material LI,Be~ EAc.ll '8c<m.... ~\'&lo.jg.c ~\ FE'~Ho;;:'NTATION ~l.~~:~c*j / ~/ C HAH"E E" R c; s"... 'PLINCO 'P~ C.,v~t> WIT.. Elsc:T&JU.L. TA?f, Setting up the experiment 1. Cut your chosen plant material, such as corn, alfalfa, grass clippings or other green plant material into 1 em pieces and then divide into two (or three) portions. You will need enough to pack your silos (approximately 500 ml for each silo). If you are using corn, include the kernels and husk only; do not attempt to cut up the cob. 2. Label the chambers as "control" and "yogurt" or "inoculant," depending on how many variables you intend to test. Also attach similar labels to outside of collector chamber at each syringe. 3. Spread 500 grams of dry material on a table. Using a small syringe, draw up 10 ml of yogurt from container. Evenly apply small drops of yogurt across sample and thoroughly mix. Pack material tightly into chamber labelled as "yogurt." Clean the table and syringe thoroughly before proceeding to next step. 4. Spread another 500 grams of the dry material on a table. Measuring with a film can lid, fill the depression in a lid with dry inoculant, and pour into film container. Using a clean syringe, draw up 10 ml of water and place it into film container. Snap on lid and shake to mix. Draw up mixture into syringe and evenly apply small drops Fermentation I 5-23

24 Part II: Student material across sample and mix thoroughly. Pack material tightly into the chamber labelled "inoculant." 5. Pack the third chamber with 500 grams of dry material. This is your control. 6. Insert tops into chambers with neck up and push down plant material compressing it very tightly. Care should be used to press out as much air as possible and to compress the samples until all chambers are even in height. 7. While holding down chamber top, have a helper apply a continuous bead of silicone around the outer edge of the chamber top, sealing it to the inside of the chamber bottom. Repeat with each chamber. 8. Poke a small hole with a hot nail poke at the mid-point of the chamber to use as a sampling port for temperature and ph. Cover with a small piece of electrical tape, ensuring a good seal. 9. Make a hypothesis and predict the results of this activity. 10. You will record your data every day for the first week, and on Monday, Wednesday, and Friday of the following week. One data point will be taken in the third week. You will measure and record the following: ph of the material-open the sampling port on the side of the bottle and insert ph paper. You may need to rub the paper around a bit to get moisture on the paper. Remove the paper and record the ph. Temperature of the material-insert a thermometer into the center of the silo through the sampling hole and record the temperature. Remove thermometer and tightly reseal the hole with a new piece of electrical tape. Presence of C0 2 -You can measure the presence of C0 2 by bubbling the C0 2 through water, cabbage juice or limewater. Volume of C0 2 -At the end of the experiment a final DM test should be done to determine the value of the final product. This assessment should only be made on silos that have stayed anaerobic. Silos that look moldy or off-color should not be opened and should be disposed of I Fermentation

25 Part II: Student material Bunk life study (Optional) Open only the chambers that have remained anaerobic and spread the fermented material out on a paper plate. The material should have a fresh acidic aroma. Fermentation I 5-25

26 Part II: Data sheets Mixing in some microbes Student name:------: Plant material used in silo Initial dry rna tter % Initial ph of material ph Temperature C0 2 produced I time Day Silo 1 Silo 2 Silo 3 Silo 1 Silo 2 Silo 3 Silo 1 Silo 2 Silo 3 Final dry matter percentage: Silo 1 =_%,silo 2 =_%,silo 3 = _% Comments or observations: 5-26 I Fermentation

27 Part II: Data sheets Graph 1: Daily ph change in mini-silos ph Time Graph 2: Daily temperature changes in mini-silos ph Time Results and discussion 1. Compare the trends in each bottle as shown on the graphs. a. What happened to the ph in each bottle? b. What happened to the temperature in each bottle? c. What was the trend in gas production in each bottle? d. Were the results different between bottles? Why? e. What was the correlation between the additive and the fermentation process? 2. What was the gas being produced in the syringe? How could you prove this? Fermentation I 5-27

28 Part II: Student material 3. How did this activity illustrate the idea of symbiosis? 4. How did your hypothesis compare with the actual results? 5. What other variables could you investigate with this activity? Extensions 1. What would be the effect on fermentation of other variables such as feed type, system temperature, compaction, and moisture? 2. What bacteria are actually present in the final product? Try plating them out on an agar medium. 3. Are there other ph indicator solutions which could be used? 4. What other variables could be tested for? 5. Is your product palatable? Try presenting it to any ruminants on your farm. 6. What other kinds of foods/feeds could we preserve? 7. How could a genetically-engineered form of bacteria be more efficient? 8. What applications could this have for long-term food preservation or space exploration? 9. Could you design a way to measure the type of acid produced or the type of bacteria produced? 10. Are there other substances that could be used as natural inoculants? 5-28 I Fermentation