FUNDAMENTALS OF MICROBIOLOGY - E. Yu. Tyumentseva - 2015

TOPIC 4. ANAEROBIC DECOMPOSITION (FERMENTATION)

Fermentation is the anaerobic (occurring in the absence of oxygen) metabolic breakdown of nutrient molecules, such as glucose. In the words of Louis Pasteur, "fermentation is life without oxygen." Most Types of fermentation are carried out by microorganisms that are obligate or facultative anaerobes.

The term "fermentation" is also used in a broader sense to refer to the rapid growth of microorganisms in a suitable medium. When used in this sense, no distinction is made between aerobic and anaerobic METABOLISM.

Fermentation is often used for food preparation or preservation. When speaking of fermentation, we typically refer to the fermentation of sugar (its conversion into alcohol) using Yeast, but, for example, Other types of fermentation are used in yogurt production.

Human Use of fermentation typically involves the application of specific species and strains of microorganisms. Wines are sometimes improved using a process of co-fermentation.

Lactic acid Bacteria are divided into two groups: homofermentative and heterofermentative. Homofermentative bacteria (e.g., Lactobacillus delbrueckii) break down Monosaccharides to form two molecules of lactic acid According to the overall equation:

C₆H₁₂O₆ = 2CH₃CHOH-COOH.

Heterofermentative bacteria (e.g., Bacterium lactis aerogenes) carry out fermentation to produce lactic acid, acetic acid, ethyl alcohol, and CO₂, as well as small amounts of aromatic substances such as diacetyl, esters, etc.

In Lactic acid fermentation, The conversion of CARBOHYDRATES, especially in the initial stages, is closely related to the reactions of Alcoholic Fermentation, except for the decarboxylation of pyruvic acid, which is reduced to lactic acid by hydrogen obtained from NADH. Homofermentative lactic acid fermentation is used to produce lactic acid, in the manufacture of various fermented dairy products and bread, and in silage production in agriculture. Heterofermentative lactic acid fermentation occurs during the preservation of various fruits and vegetables by pickling.

Lactic acid fermentation is The breakdown of sugar by lactic acid bacteria to form lactic acid. In its overall summary form, it can be represented by the following equation:

С₆Н1₂О6 = 2С₃Н₆О₃ + 18 kcal.

This fermentation is commonly observed in milk, causing it to sour. This is where the type of fermentation, the bacteria that cause it, and the main product of fermentation—the acid—get their names. Lactic acid bacteria can be spherical or rod-shaped. They are non-motile, do not form spores, and are facultative anaerobes.

Under identical conditions, different species of lactic acid bacteria produce varying amounts of acid, which is due to their differing acid tolerance. Rod-shaped bacteria produce more acid than spherical ones (cocci).

Lactic acid bacteria are capable of fermenting only mono- and Disaccharides, and do not ferment Starch and other Polysaccharides at all because they do not secrete the corresponding Enzymes. Some of these bacteria produce antibiotic substances that act against PATHOGENS OF INTESTINAL diseases.

Lactic acid bacteria are widely distributed in nature; they are constantly found in soil, on various plants, on fruits and vegetables, in milk, etc.

The most important lactic acid bacteria include: lactic streptococcus, Bulgarian, acidophilus, cheese, Delbrueck, cucumber, and cabbage bacilli, among others.

Lactic streptococcus consists of spherical bacteria arranged in pairs or short chains. They grow best at temperatures of 30–35 °C, with a minimum Temperature limit of around 10 °C. During fermentation, they accumulate up to 1% acid. They are widely used in the preparation of fermented dairy products (sour milk, kefir, sour cream, cottage cheese, etc.).

The Bulgarian bacillus often forms long chains and was originally isolated from Bulgarian sour milk. It is a non-motile, non-spore-forming rod. The optimal temperature for its growth is 40–45 °C, with a minimum temperature of 20 °C. It produces up to 3.5% lactic acid in milk.

The acidophilus bacillus was isolated from the intestinal excretions of an infant. It has an optimal temperature of about 40 °C and a minimum growth temperature of 20 °C. It accumulates up to 2.2% lactic acid in milk. It is used to prepare fermented dairy products such as acidophilin and acidophilus milk.

The cheese bacillus has an optimal temperature of about 40 °C and is used in cheesemaking.

Delbrueck's bacillus consists of single or short-chained Cells that do not form spores. Its optimal temperature is 45 °C. It produces up to 2.5% acid in the medium. It is used for the industrial production of lactic acid, as well as in the manufacture of sourdough starters.

Cucumber and cabbage bacilli develop during the fermentation of vegetables. Lactic acid fermentation is of great industrial importance. It is used in The production of fermented dairy products, in baking, in the processes of vegetable pickling and fodder ensiling, in the manufacture of kvass, in the production of lactic acid, etc.

Lactic acid fermentation in baking helps prevent the growth of harmful bacteria in dough that cause rope spoilage (ropiness) in bread, and also helps improve the flavor profile of the bread.

The lactic acid produced As a result of this fermentation imparts a distinctive flavor to fermented vegetables and prevents the growth of putrefactive bacteria.

During Butyric acid fermentation, sugars are broken down by bacteria under anaerobic conditions, yielding butyric acid, carbon dioxide, and hydrogen. The reaction proceeds according to the equation:

С₆Н₁₂О₆ = С₃Н₇СООН + 2СО₂ + 2Н₂ + 20 kcal.

By-products of this process include ethyl and butyl alcohols, acetic acid, etc. Such fermentation can occur in milk and dairy products, imparting an unpleasant taste and odor characteristic of butyric acid. The butyric acid bacteria that cause this fermentation are peritrichously flagellated, motile, spore-forming rods, with an optimal growth temperature ranging between 30 and 40 °C. They are obligate anaerobes and can only reproduce in the complete absence of atmospheric oxygen or at very low concentrations. The spores formed by butyric acid bacteria are highly resistant to adverse conditions, withstanding boiling for several minutes and dying only after prolonged sterilization. They are located either centrally or closer to one of The Cell ends, giving the cell a spindle or tennis racket shape.

Butyric acid bacteria are capable of fermenting both simple sugars and more complex carbohydrates, such as starch, pectin substances, and others, as well as glycerol. These bacteria are widely distributed in nature, occurring in soil, the silt of lakes, ponds, and marshes, in accumulations of various residues and waste, manure, contaminated Water, milk, cheese, etc. The Fermentation caused by these bacteria plays an important role in the transformation of matter in nature.

In the national economy, butyric acid fermentation can cause significant harm, as butyric acid bacteria can lead to the mass spoilage of potatoes and vegetables, rancidity of milk, late blowing of cheeses, spoilage of canned food, etc.

Butyric acid bacteria are inhibited by an acidic environment; therefore, where lactic acid bacteria develop and produce lactic acid, The activity of butyric acid bacteria is suspended. However, if lactic acid accumulates slowly in fermented vegetables, they can spoil due to the proliferation of butyric acid bacteria. These bacteria cause the spoilage of pasteurized milk, where lactic acid fermentation is excluded, as well as raw milk during long-term cold storage when the activity of lactic acid bacteria is weakened.

Developing in damp flour, butyric acid bacteria give it a rancid taste. Butyric acid fermentation has Structure/179.html">Practical Applications in the production of butyric acid, which is widely used in industry.

Laboratory Procedure

Objective: to study the fermentation process as a result of the vital activity of lactic acid and butyric acid bacteria.

Materials, Reagents, Equipment: Microscope; bacteriological loops and pipettes; cover slips and microscope slides; alcohol burner; incubator; cambric napkin; test tube rack; filter paper; methylene blue; immersion oil; benzine; milk; fermented milk product; unpeeled potatoes; chalk; Lugol's solution; alcohol-ether mixture (1:1); 5% FеСl₃ solution; ethyl alcohol; concentrated sulfuric acid.

Experiment No. 1. Isolation of Microorganisms of the Lactic Acid Fermentation Process

Fermented milk products can serve as an inoculum for obtaining enrichment cultures of lactic acid bacteria.

Sterile milk is used as a growth medium. Add 1 mL of sour milk or another fermented dairy product to a test tube containing sterile milk. Place the inoculated tube and a control tube (with sterile milk) in an incubator at 30–32 °C for 1–2 days (or at 20–25 °C for a week). Then, observe the growth CHARACTERISTICS OF THE microorganisms based on milk coagulation, noting clot formation (dense, loose, slimy) and gas production. Next, perform Microscopy on a fixed cell preparation made from the culture liquid. Note the morphological Features of the bacterial cells: shape, spore formation, motility, etc.

Given The Nature of the substrate (milk), the fixed cell preparation is prepared as follows: using a sterile bacteriological loop, place a drop of the culture liquid onto a degreased microscope slide and spread it evenly with a cover slip. Air-dry the smear, then fix it (hold the slide smear-side up with forceps and pass it through the burner flame three times) and simultaneously degrease it for 10 minutes with an alcohol-ether mixture (1:1) applied directly to the smear. After evaporation, apply the mixture a second time. Stain the dried smear with methylene blue (place the fixed preparation on parallel Glass rods; apply the dye solution from a pipette onto the slide; the staining time ranges from 1 to 3 minutes, during which the dye must cover the entire smear, then rinse it with a gentle stream of water to wash away any dye not adsorbed by the microorganisms; finally, dry the preparation by gently blotting it with filter paper).

Experiment No. 2. Isolation of Microorganisms of the Butyric Acid Fermentation Process

To obtain an enrichment culture of butyric acid bacteria, place several pieces of unpeeled potato and a quarter teaspoon of chalk into a large 50 mL test tube, fill it with tap water (leaving a 2–3 cm gap to the plug), seal it with a cotton plug, and pasteurize it in a water bath at 80 °C for 10 minutes, then place it in an incubator at 37 °C.

After 1–2 days, microscopy of the liquid at the bottom of the test tube reveals A large number of motile rods. A distinctive feature of butyric acid bacteria is their ability to accumulate a reserve substance, granulose, within their cells, as well as to form spores. During sporulation, the cells swell either in the middle or at the end. After the spores mature, the granulose disappears from the cells.

After 5–6 days of cultivation, analyze the enrichment culture. To prepare a microscopic slide, use a pipette to take the culture liquid containing microorganisms from the bottom of the test tube, near the potato slices.

To detect butyric acid in the culture liquid, perform two qualitative reactions.

1. Transfer 3–5 mL of the culture liquid, taken from the middle of the enrichment culture tube, into a clean, dry test tube. Add 1–2 mL of a 5% ferric chloride solution. Warm gently. Observe the appearance of a brick-brown coloration due to The formation of ferric butyrate.

2. Transfer 3–5 mL of the culture liquid, taken from the middle of the enrichment culture tube, into a clean, dry test tube. Add 1–2 mL of 96% ethyl alcohol and 1 mL of concentrated sulfuric acid. After cooling the test tube, detect the pineapple odor of the formed ethyl butyrate.

To detect granulose in the cells of butyric acid bacteria, apply a drop of concentrated Lugol's solution to a drop of the culture liquid containing bacteria for 5–10 minutes, then cover with a cover slip and examine under a microscope. Granulose stains dark brown.

Recording and Analyzing Research Results

1. Obtain an enrichment culture of lactic acid bacteria from kefir or sour milk, and describe the growth characteristics. Prepare a fixed cell slide and examine it under a microscope (90x objective). Observe and draw the microscopic views of the studied cultures. Label the magnification of the preparation under each drawing.

2. Obtain an enrichment culture of butyric acid bacteria, and describe the growth characteristics. Prepare a fixed cell slide and examine it under a microscope (90x objective). Observe and draw the microscopic views of the studied cultures. Label the magnification of the preparation under each drawing.

While studying this topic, students will learn how to obtain enrichment cultures of lactic acid and butyric acid bacteria, and will reinforce the microbiological Research Methods essential for their professional activities.

Review Questions

1. What is lactic acid fermentation, and what is its significance for lactic acid bacteria?

2. What shape do lactic acid bacteria cells have?

3. What type of Nutrition and Respiration do lactic acid bacteria exhibit?

4. Is the optimum growth temperature the same for different lactic acid bacteria?

5. Which experimental conditions favor the enrichment of lactic acid bacteria, and what limits the growth of contaminating microflora?

6. What are the PRACTICAL APPLICATIONS OF lactic acid bacteria?

7. What is butyric acid fermentation, and what is its significance for butyric acid bacteria?

8. What shape do butyric acid bacteria cells have, and do they form spores?

9. What type of nutrition and respiration do butyric acid bacteria exhibit?

10. How can the presence of butyric acid bacteria and their metabolic products be detected in the experiment? Support your answer with chemical equations.

11. Which experimental conditions favor the enrichment of butyric acid bacteria?

12. What are the practical applications of butyric acid bacteria?

13. What other types of fermentation do you know? What is the Biological Significance of fermentation for the life processes of microorganisms?