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6: Oxygen Requirements and Anaerobes - Biology

6: Oxygen Requirements and Anaerobes - Biology


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Learning Objectives

  • Identify the 3 major categories of microbes based on oxygen requirements.
  • Learn different ways to culture anaerobic bacteria.

HOW TO DETERMINE OXYGEN REQUIREMENTS

An excellent way to determine the oxygen needs of your bacterium is to grow it in different oxygen environments---atmospheric oxygen of 22%, no oxygen at all (GasPak jar), and reduced oxygen at less than 10% (candle jar)--and compare the quality and quantity of growth.

TYPES OF OXYGEN ENVIRONMENTS

The candle jar at right has 3-5% CO2 and 8-10% O2 (0.3% and 21% in the atmosphere, respectively). This is a handy way to determine if you have an aerobe which is microaerophilic, since they grow optimally under reduced (but present) oxygen conditions as in the candle jar. Many microaerophilic bacteria will grow poorly at 22% O2, whereas some will not grow at all (e.g. Neisseria gonorrhoea). Possibly the by-products of aerobic respiration, superoxide radicals and hydrogen peroxide, make it difficult for the microaerophiles to do well in 22% O2. Some microaerophiles are actually capnophilic (requiring elevated CO2 levels to grow). Strict aerobes may not grow well in a candle jar, depending on the species. The Gram+ genus Bacillus and Gram– genus Pseudomonas include aerobic bacillus-shaped bacteria.

On the left is a GasPak jar, with a gas generator envelope inside. The environment is 0% O2,.

The newer anaerobic system (seen at right) consists of a plastic container (for the agar plates) and a paper gas generating sachet. The sachet contains ascorbic acid and activated carbon which reacts on exposure to air, when removed from the enclosed envelope. Oxygen is rapidly absorbed and CO2 is produced. When the paper sachet is placed in a sealed plastic pouch, this reaction will create ideal atmospheric conditions for the growth of anaerobes—anaerobic within 2.5 hours.
Because a GasPak jar looks the same, whether it has oxygen inside or not, an indicator strip, containing methylene, is included in the jar. Methylene blue is blue when oxidized, colorless when reduced. The carbon within the pouch reacts with free oxygen in the jar, producing 10-15% CO2.

Quite a few human pathogens are strict anaerobes, exemplified by the bacillus-shaped genera---Gram– Bacteroides, Bacillus (anthracis), and Gram+ Clostridium(tetani, botulinum).

Aerotolerants are anaerobes that can grow in the presence of O2 (compared to the strict anaerobes which would likely die), but they do not use it. And last, but very common, are the facultative anaerobes which prefer to use O2 when present but will grow without it.


Another way to culture and grow anaerobes is the use of reduced media--media without oxygen. Thioglycollate broth has a reducing agent in it---the chemical thioglycollate---which binds any free oxygen within the medium. You will also notice that these tubes have screw caps, allowing a tight closure, to reduce oxygen entry. However, some oxygen will be in the tube between the cap and the broth and there is no way to get rid of it. So there will be some diffusion of oxygen into the top portion of the broth, and that is where any aerobic bacteria may grow. An indicator, resazurin, in the medium will be a light pink in the area of higher oxygen. You can determine whether the bacterium is an anaerobe, facultative anaerobe, or an aerobe by checking out where the organism grows in the column of media. DO NOT SHAKE IT!

Note

Oxygen will permeate the broth when this medium sits around for a while.

Check for the pink color: if so, boil the broth for 5 minutes (removes the oxygen).

growth is indicated by gray area

MATERIALS NEEDED: per table

  • 1 thioglycollate broth per table
  • 3 TSA plates (divide into pie-shaped sections)
  • GasPak container for entire lab + GasPak sachet for the jar + methylene blue indicator strip candle jar for entire lab
  • cultures
    • your table’s unknown bacterium
    • a strict aerobe + a strict anaerobe used as controls

Note

Your instructor will give you the names at beginning of lab

Note

Your instructor will set up the strict aerobe and the strict anaerobe cultures in thioglycollates for the class to view.

THE PROCEDURES

Thioglycollate broth

  1. The thioglycollate broth should be either boiled first before inoculation OR recently made so that the oxygen content is very low. (Your instructor will tell you if it needs to be boiled).
  2. Inoculate a tube of thioglycollate broth with your unknown bacterium: make sure that the loop or needle goes down to the BOTTOM of the broth (do not get metal holder in the sterile broth).
  3. Incubate at 25 or 37 degrees C as directed.

TSA plates in 3 different oxygen environments

  1. Label 3 plates for the table---candle jar, ambient air, and GasPak anaerobic jar.
  2. Divide the 3 plates into sections, one for each organism—your unknown, the strict aerobe, the strict anaerobe.
  3. Inoculate the section by streaking a straight line or a zigzag (as seen below). HOWEVER, be sure that you inoculate all 3 plates using the same technique.
  4. Be sure that the jar has a methylene blue indicator strip (seen above) inside. The methylene blue is blue when oxidized, but colorless when reduced. Before the jar is opened, the strip should be checked to make sure that it is COLORLESS.
  5. Incubate at 30 or 37 degrees C

INTERPRETATION: after incubation

TSA plates

Compare the presence/absence of growth, as well as the quantity of growth on the 3 plates. Determine whether aerobic, anaerobic, or facultatively anaerobic.

To the right:

  • A is an facultative anaerobe
  • B is an aerobe (microaerophilic)
  • C is an anaerobe

Thioglycollate broth

Determine WHERE the most amount of growth occurs in the column of liquid---the top, the bottom, top to bottom. DO NOT SHAKE IT! Can you determine if the bacterium is aerobic, anaerobic, or facultatively anaerobic?

QUESTIONS

  1. Why should you boil thioglycollate broth if it is not freshly made?
  2. Which environment would a microaerophilic bacterium like the best?
  3. Data from TSA plates in different environments. Record as -, +1, or +2 growth.

WRITE IN NAMES OF BACTERIA.

  1. Data from thioglycollate broths. Shade in where the growth is located for the 3 bacteria.

LABEL WITH NAMES OF BACTERIA.


6: Oxygen Requirements and Anaerobes - Biology

Article Summary:

Oxygen requirements of different bacteria

Almost all plants and animals are dependent upon supply of atmospheric oxygen which is unlikely in bacteria. Depending upon the oxygen need in bacteria, they are classified into 4 groups: strict or obligate aerobic that grow only in the presence of oxygen. Facultative anaerobic bacteria grow both in presence and absence of free oxygen. Some bacteria are microaerophilic which grow best in the presence of low concentration of molecular oxygen. However, not all bacteria require oxygen for growth. Strict or obligate anaerobic bacteria can grow only in the absence of oxygen.

Amount of oxygen required is different for growth and for other metabolic activities. Aerobic bacteria requires large surface growth area and exposure to contact available atmospheric oxygen, therefore under in vitro conditions, they are grown on shallow agar plates. Broth culturing of such bacteria is always accompanied by shaking and bubbling by spargers or baffles. Both of these mechanical actions are important during fermentation reactions to increase the availability and consumption of oxygen to growing aerobes. For the cultivation of anaerobic bacteria special techniques are employed which are meant to exclude atmospheric oxygen from growth medium. For this purpose, prereduced growth media contained with reducing agents like thioglycollate, formaldehyde, and sulfoxalate or cysteine hydrochloride which effectively absorb molecular oxygen are employed. Mechanical removal of oxygen by various means from an enclosed vessel containing tubes or plates of inoculated medium is another option. In one method, air is pumped out of vessel and replaced by gases like N2, helium or mixture of CO2 and N2. Burning of a candle to utilize oxygen present in the growth vessel is also one of the simplest ways to create oxygen free atmosphere. Addition of pyrogallol over the plug followed by rubber corking of test tube is used for slant cultivation of anaerobic bacteria. Anaerobic gaspak jars are routinely employed in various laboratories to cultivate anaerobes like Clostridium, Bacteroids and microaerophilic bacteria like Borrelia, Streptococci or Campylobacter.

Role of oxygen: Oxygen is elemental constituent of water and organic compounds. Obligatory aerobic bacteria are dependent on aerobic respiration for fulfillment of their energetic needs wherein molecular oxygen functions as terminal electron acceptor or oxidising agent. Anaerobic bacteria do not obtain energy by using molecular oxygen. In metabolic terms, facultative anaerobic bacteria can use oxygen as terminal oxidising agent only when available but can also obtain energy in its absence by fermentative reactions such as that in all enterobacteria. Some of them are not sensitive to presence of oxygen and hence have exclusively fermentative energy yielding metabolism lactic acid bacteria are representative of such fermentative metabolism. Microaerophilic bacteria grow best at oxygen concentration of 0.2atm pressure and possess enzymes that are inactivated during strong oxidising conditions, hence functional only at low oxygen pressure. Oxygen is also co-substrate for oxygenase enzymes that catalyze degradation of aromatic and alkane compounds. Oxygenative cleavage of aromatics is very useful for their dissimilation. Oxidative dissimilation of recalcitrant pollutant aromatics is hence one of the potential requirements for their efficient biodegradation. Oxygenases also mediate sterol and unsaturated fatty acid synthesis. They catalyze direct addition of one or two oxygen atoms to organic substrate compounds.

Oxygen toxicity: The role of nodulation in nitrogen fixing species of Rhizobium strains was understood only when researchers came to know about oxygen toxicity. Biological nitrogen fixation is catalyzed by nitrogenase enzyme system which is very sensitive to the presence of oxygen. It is readily inactivated by presence of molecular oxygen halting the vital process of fixation of atmospheric nitrogen. Similarly, hydrogen utilizing reactions catalyzed by enzyme hydrogenase accompanying the nitrogen fixation are also inhibited by oxygen. If oxygen is found at high concentration greater than atmosphere it can be toxic to aerobic bacteria oxygenases of aerobes are irreversibly denatured by exposure to oxygen. Therefore, bacteria especially aerobic, possessing oxygen sensitive enzymes have developed special mechanisms to protect functional enzymes from oxygen inactivation. Some mechanisms include high respiration rate, heterocyst formation, and synthesis of exopolysaccharides or nodulation. Enzymes like superoxide dismutase (SOD), peroxidase, and catalase are also used by these bacteria as shield against toxic forms of oxygen like superoxide radical (O2.-), hydrogen peroxide (H2O2) and hydroxyl radical (OH.). Lactic acid bacteria that don't possess catalase degrade H2O2 by peroxidase to H2O. Lethal accumulation of superoxide is prevented by SOD which catalyses its conversion to O2 and H2O2. Oxidations of flavoproteins by O2 results in the formation of toxic compounds like superoxide radicals. Oxygen is also toxic to anaerobic bacteria as they do not contain SOD (very little if present) or catalase therefore they have adapted to fermentative metabolism whereby they avoid presence of oxygen. Oxygen in its nascent or singlet state (1O2) is very toxic and powerful oxidant. Toxic singlet state is generated when triplet state of photosensitizer reacts with oxygen during photo-oxidation, a process similar to the production of superoxide radical. Its toxicity is enhanced in presence of light and photosensitive pigments or photosensitizers. Carotenoid pigments quench this form of oxygen and protect the cell from photo oxidative death. The chlorophylls are powerful photosensitizers and hence carotenoids are always present alongwith chlorophylls. Nonphotosynthetic aerobic bacteria like Micrococcus and Serratia also produce carotenoids in cell membrane to nullify the effect of photosensitizer cytochromes during their growth in high light intensified environment. Knowledge of oxygen requirements of bacteria is critical factor not only for their cultivation and classification but it also represents ecological significance.

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Facultative Anaerobes

Bacteria described as facultative anaerobes grow well in oxygen but can also continue growing in its absence. Although they particularly thrive in the presence of oxygen, these anaerobes can also use such processes as fermentation to continue growing when oxygen is not available.

Therefore, facultative anaerobes may be described as having the following three main characteristics:

They can either grow aerobically or anaerobically

Because of their ability to respire and ferment organic substances, these types of bacteria (facultative anaerobes), can continue growing in the presence or the absence of oxygen. For some of these organisms, particularly those that rely on oxygen for some of the biosynthetic reactions, growing is significantly affected in the absence of oxygen.


CLASSIFICATION BASED ON OXYGEN REQUIREMENT

  1. Strict Aerobes: These do not grow in the absence of oxygen.
  2. Anaerobes: These can be two types:
  3. Strict (Obligatory) anaerobes: Bacteria that can grow only in the absence of oxygen.
  4. Facultative anaerobes: These can grow both in presence or absence of oxygen. Most of the commonly isolated bacteria belong to this group.
  5. Carboxyohillic: These require presence of high percentage (10%) of carbon dioxide.
  6. Microaerophilic: These require only small amount of oxygen for their growth and higher concentration of the oxygen will kill the organism.

pH is the measure of acidity or alkalinity of an environment on a scale that has neutral at pH7. Environments that are acidic have pH values below 7 those that are alkaline have pH values above 7. Most microorganisms grow best at close to the neutral pH value (pH 6.6 to 7.5). Only a few microorganisms grow in very acid conditions below a pH of 4.0. Bacteria grow at a fairly specific pH for each species, but fungi grow over a wider range of pH values. Microbes can be classified based hydrogen potential as follows

Acidophiles: have growth optimum between pH 0 and 5.5 e.g. Sulfolobus, Picrophilus
Neutrophiles: have growth optimum between pH 5.5 and 8.0 e.g. Escherichia, Euglena
Alkaliphiles (alkalophiles): have growth optimum between pH 8.0 and 11.5 e.g. Bacillus acidophilus
Extreme alkaliphiles: have growth optima at pH 10 or higher


9.2 Oxygen Requirements for Microbial Growth

Ask most people “What are the major requirements for life?” and the answers are likely to include water and oxygen. Few would argue about the need for water, but what about oxygen? Can there be life without oxygen?

The answer is that molecular oxygen (O2) is not always needed. The earliest signs of life are dated to a period when conditions on earth were highly reducing and free oxygen gas was essentially nonexistent. Only after cyanobacteria started releasing oxygen as a byproduct of photosynthesis and the capacity of iron in the oceans for taking up oxygen was exhausted did oxygen levels increase in the atmosphere. This event, often referred to as the Great Oxygenation Event or the Oxygen Revolution , caused a massive extinction. Most organisms could not survive the powerful oxidative properties of reactive oxygen species (ROS), highly unstable ions and molecules derived from partial reduction of oxygen that can damage virtually any macromolecule or structure with which they come in contact. Singlet oxygen (O2•), superoxide ( O 2 − ) , ( O 2 − ) , peroxides (H2O2), hydroxyl radical (OH•), and hypochlorite ion (OCl − ), the active ingredient of household bleach, are all examples of ROS. The organisms that were able to detoxify reactive oxygen species harnessed the high electronegativity of oxygen to produce free energy for their metabolism and thrived in the new environment.

Oxygen Requirements of Microorganisms

Many ecosystems are still free of molecular oxygen. Some are found in extreme locations, such as deep in the ocean or in earth’s crust others are part of our everyday landscape, such as marshes, bogs, and sewers. Within the bodies of humans and other animals, regions with little or no oxygen provide an anaerobic environment for microorganisms. (Figure 9.19).

We can easily observe different requirements for molecular oxygen by growing bacteria in thioglycolate tube culture s. A test-tube culture starts with autoclaved thioglycolate medium containing a low percentage of agar to allow motile bacteria to move throughout the medium. Thioglycolate has strong reducing properties and autoclaving flushes out most of the oxygen. The tubes are inoculated with the bacterial cultures to be tested and incubated at an appropriate temperature. Over time, oxygen slowly diffuses throughout the thioglycolate tube culture from the top. Bacterial density increases in the area where oxygen concentration is best suited for the growth of that particular organism.

The growth of bacteria with varying oxygen requirements in thioglycolate tubes is illustrated in Figure 9.20. In tube A, all the growth is seen at the top of the tube. The bacteria are obligate (strict) aerobe s that cannot grow without an abundant supply of oxygen. Tube B looks like the opposite of tube A. Bacteria grow at the bottom of tube B. Those are obligate anaerobe s, which are killed by oxygen. Tube C shows heavy growth at the top of the tube and growth throughout the tube, a typical result with facultative anaerobe s. Facultative anaerobes are organisms that thrive in the presence of oxygen but also grow in its absence by relying on fermentation or anaerobic respiration, if there is a suitable electron acceptor other than oxygen and the organism is able to perform anaerobic respiration. The aerotolerant anaerobe s in tube D are indifferent to the presence of oxygen. They do not use oxygen because they usually have a fermentative metabolism, but they are not harmed by the presence of oxygen as obligate anaerobes are. Tube E on the right shows a “Goldilocks” culture. The oxygen level has to be just right for growth, not too much and not too little. These microaerophile s are bacteria that require a minimum level of oxygen for growth, about 1%–10%, well below the 21% found in the atmosphere.

Examples of obligate aerobes are Mycobacterium tuberculosis , the causative agent of tuberculosis and Micrococcus luteus , a gram-positive bacterium that colonizes the skin. Neisseria meningitidis , the causative agent of severe bacterial meningitis , and N. gonorrhoeae, the causative agent of sexually transmitted gonorrhea , are also obligate aerobes.

Many obligate anaerobes are found in the environment where anaerobic conditions exist, such as in deep sediments of soil, still waters, and at the bottom of the deep ocean where there is no photosynthetic life. Anaerobic conditions also exist naturally in the intestinal tract of animals. Obligate anaerobes, mainly Bacteroidetes , represent a large fraction of the microbes in the human gut. Transient anaerobic conditions exist when tissues are not supplied with blood circulation they die and become an ideal breeding ground for obligate anaerobes. Another type of obligate anaerobe encountered in the human body is the gram-positive, rod-shaped Clostridium spp. Their ability to form endospores allows them to survive in the presence of oxygen. One of the major causes of health-acquired infections is C. difficile, known as C. diff. Prolonged use of antibiotics for other infections increases the probability of a patient developing a secondary C. difficile infection. Antibiotic treatment disrupts the balance of microorganisms in the intestine and allows the colonization of the gut by C. difficile, causing a significant inflammation of the colon.

Other clostridia responsible for serious infections include C. tetani, the agent of tetanus, and C. perfringens, which causes gas gangrene . In both cases, the infection starts in necrotic tissue (dead tissue that is not supplied with oxygen by blood circulation). This is the reason that deep puncture wounds are associated with tetanus. When tissue death is accompanied by lack of circulation, gangrene is always a danger.

The study of obligate anaerobes requires special equipment. Obligate anaerobic bacteria must be grown under conditions devoid of oxygen. The most common approach is culture in an anaerobic jar (Figure 9.21). Anaerobic jars include chemical packs that remove oxygen and release carbon dioxide (CO2). An anaerobic chamber is an enclosed box from which all oxygen is removed. Gloves sealed to openings in the box allow handling of the cultures without exposing the culture to air (Figure 9.21).

Staphylococci and Enterobacteriaceae are examples of facultative anaerobes. Staphylococci are found on the skin and upper respiratory tract. Enterobacteriaceae are found primarily in the gut and upper respiratory tract but can sometimes spread to the urinary tract, where they are capable of causing infections. It is not unusual to see mixed bacterial infections in which the facultative anaerobes use up the oxygen, creating an environment for the obligate anaerobes to flourish.

Examples of aerotolerant anaerobes include lactobacilli and streptococci, both found in the oral microbiota. Campylobacter jejuni , which causes gastrointestinal infections, is an example of a microaerophile and is grown under low-oxygen conditions.

The optimum oxygen concentration , as the name implies, is the ideal concentration of oxygen for a particular microorganism. The lowest concentration of oxygen that allows growth is called the minimum permissive oxygen concentration . The highest tolerated concentration of oxygen is the maximum permissive oxygen concentration . The organism will not grow outside the range of oxygen levels found between the minimum and maximum permissive oxygen concentrations.

Check Your Understanding

  • Would you expect the oldest bacterial lineages to be aerobic or anaerobic?
  • Which bacteria grow at the top of a thioglycolate tube, and which grow at the bottom of the tube?

Case in Point

An Unwelcome Anaerobe

Charles is a retired bus driver who developed type 2 diabetes over 10 years ago. Since his retirement, his lifestyle has become very sedentary and he has put on a substantial amount of weight. Although he has felt tingling and numbness in his left foot for a while, he has not been worried because he thought his foot was simply “falling asleep.” Recently, a scratch on his foot does not seem to be healing and is becoming increasingly ugly. Because the sore did not bother him much, Charles figured it could not be serious until his daughter noticed a purplish discoloration spreading on the skin and oozing (Figure 9.22). When he was finally seen by his physician, Charles was rushed to the operating room. His open sore, or ulcer, is the result of a diabetic foot .

The concern here is that gas gangrene may have taken hold in the dead tissue. The most likely agent of gas gangrene is Clostridium perfringens , an endospore-forming, gram-positive bacterium. It is an obligate anaerobe that grows in tissue devoid of oxygen. Since dead tissue is no longer supplied with oxygen by the circulatory system, the dead tissue provides pockets of ideal environment for the growth of C. perfringens.

A surgeon examines the ulcer and radiographs of Charles’s foot and determines that the bone is not yet infected. The wound will have to be surgically debrided (debridement refers to the removal of dead and infected tissue) and a sample sent for microbiological lab analysis, but Charles will not have to have his foot amputated. Many diabetic patients are not so lucky. In 2008, nearly 70,000 diabetic patients in the United States lost a foot or limb to amputation, according to statistics from the Centers for Disease Control and Prevention. 1

Detoxification of Reactive Oxygen Species

Aerobic respiration constantly generates reactive oxygen species (ROS), byproducts that must be detoxified. Even organisms that do not use aerobic respiration need some way to break down some of the ROS that may form from atmospheric oxygen. Three main enzymes break down those toxic byproducts: superoxide dismutase, peroxidase, and catalase. Each one catalyzes a different reaction. Reactions of type seen in Reaction 1 are catalyzed by peroxidase s.

In these reactions, an electron donor (reduced compound e.g., reduced nicotinamide adenine dinucleotide [NADH]) oxidizes hydrogen peroxide , or other peroxides, to water. The enzymes play an important role by limiting the damage caused by peroxidation of membrane lipids. Reaction 2 is mediated by the enzyme superoxide dismutase (SOD) and breaks down the powerful superoxide anions generated by aerobic metabolism:

The enzyme catalase converts hydrogen peroxide to water and oxygen as shown in Reaction 3.

Obligate anaerobes usually lack all three enzymes. Aerotolerant anaerobes do have SOD but no catalase. Reaction 3, shown occurring in Figure 9.23, is the basis of a useful and rapid test to distinguish streptococci, which are aerotolerant and do not possess catalase, from staphylococci, which are facultative anaerobes. A sample of culture rapidly mixed in a drop of 3% hydrogen peroxide will release bubbles if the culture is catalase positive.

Bacteria that grow best in a higher concentration of CO2 and a lower concentration of oxygen than present in the atmosphere are called capnophiles . One common approach to grow capnophiles is to use a candle jar . A candle jar consists of a jar with a tight-fitting lid that can accommodate the cultures and a candle. After the cultures are added to the jar, the candle is lit and the lid closed. As the candle burns, it consumes most of the oxygen present and releases CO2.

Check Your Understanding

  • What substance is added to a sample to detect catalase?
  • What is the function of the candle in a candle jar?

Clinical Focus

Part 2

The health-care provider who saw Jeni was concerned primarily because of her pregnancy. Her condition enhances the risk for infections and makes her more vulnerable to those infections. The immune system is downregulated during pregnancy, and pathogens that cross the placenta can be very dangerous for the fetus. A note on the provider’s order to the microbiology lab mentions a suspicion of infection by Listeria monocytogenes , based on the signs and symptoms exhibited by the patient.

Jeni’s blood samples are streaked directly on sheep blood agar , a medium containing tryptic soy agar enriched with 5% sheep blood. (Blood is considered sterile therefore, competing microorganisms are not expected in the medium.) The inoculated plates are incubated at 37 °C for 24 to 48 hours. Small grayish colonies surrounded by a clear zone emerge. Such colonies are typical of Listeria and other pathogens such as streptococci the clear zone surrounding the colonies indicates complete lysis of blood in the medium, referred to as beta-hemolysis (Figure 9.24). When tested for the presence of catalase, the colonies give a positive response, eliminating Streptococcus as a possible cause. Furthermore, a Gram stain shows short gram-positive bacilli. Cells from a broth culture grown at room temperature displayed the tumbling motility characteristic of Listeria (Figure 9.24). All of these clues lead the lab to positively confirm the presence of Listeria in Jeni’s blood samples.

Jump to the next Clinical Focus box. Go back to the previous Clinical Focus box.


Lactic Acid Fermentation

The fermentation method used by animals and certain bacteria, like those in yogurt, is lactic acid fermentation (Figure 2). This type of fermentation is used routinely in mammalian red blood cells and in skeletal muscle that has an insufficient oxygen supply to allow aerobic respiration to continue (that is, in muscles used to the point of fatigue). In muscles, lactic acid accumulation must be removed by the blood circulation and the lactate brought to the liver for further metabolism. The chemical reactions of lactic acid fermentation are the following:

The enzyme used in this reaction is lactate dehydrogenase (LDH). The reaction can proceed in either direction, but the reaction from left to right is inhibited by acidic conditions. Such lactic acid accumulation was once believed to cause muscle stiffness, fatigue, and soreness, although more recent research disputes this hypothesis. Once the lactic acid has been removed from the muscle and circulated to the liver, it can be reconverted into pyruvic acid and further catabolized for energy.

Practice Question

Figure 2. Lactic acid fermentation is common in muscle cells that have run out of oxygen.

Tremetol, a metabolic poison found in the white snake root plant, prevents the metabolism of lactate. When cows eat this plant, it is concentrated in the milk they produce. Humans who consume the milk become ill. Symptoms of this disease, which include vomiting, abdominal pain, and tremors, become worse after exercise. Why do you think this is the case?


Bacterial Growth and Division

A population of bacteria in a liquid medium is referred to as a culture. In the laboratory, where growth conditions of temperature, light intensity, and nutrients can be made ideal for the bacteria, measurements of the number of living bacteria typically reveals four stages, or phases, of growth, with respect to time. Initially, the number of bacteria in the population is low. Often the bacteria are also adapting to the environment. This represents the lag phase of growth. Depending on the health of the bacteria, the lag phase may be short or long. The latter occurs if the bacteria are damaged or have just been recovered from deep-freeze storage.

After the lag phase, the numbers of living bacteria rapidly increases. Typically, the increase is exponential. That is, the population keeps doubling in number at the same rate. This is called the log or logarithmic phase of culture growth, and is the time when the bacteria are growing and dividing at their maximum speed.

The explosive growth of bacteria cannot continue forever in the closed conditions of a flask of growth medium. Nutrients begin to become depleted, the amount of oxygen becomes reduced, and the pH changes, and toxic waste products of metabolic activity begin to accumulate. The bacteria respond to these changes in a variety of ways to do with their structure and activity of genes. With respect to bacteria numbers, the increase in the population stops and the number of living bacteria plateaus. This plateau period is called the stationary phase. Here, the number of bacteria growing and dividing is equaled by the number of bacteria that are dying.

Finally, as conditions in the culture continue to deteriorate, the proportion of the population that is dying becomes dominant. The number of living bacteria declines sharply over time in what is called the death or decline phase.

Bacteria growing as colonies on a solid growth medium also exhibit these growth phases in different regions of a colony. For example, the bacteria buried in the oldest part of the colony are often in the stationary or death phase, while the bacteria at the periphery of the colony are in the actively-dividing lo phase of growth.

Culturing of bacteria is possible such that fresh growth medium can be added at a rate equal to the rate at which culture is removed. The rate at which the bacteria grow is dependent on the rate of addition of the fresh medium. Bacteria can be tailored to grow relatively slow or fast and, if the set-up is carefully maintained, can be maintained for a long time.

Bacterial growth requires the presence of environmental factors. For example, if a bacterium uses organic carbon for energy and structure (chemoheterotrophic bacteria) then sources of carbon are needed. Such sources include simple sugars (glucose and fructose are two examples). Nitrogen is needed to make amino acids, proteins, lipids and other components. Sulphur and phosphorus are also needed for the manufacture of bacterial components. Other elements, such as potassium, calcium, magnesium, iron, manganese, cobalt and zinc are necessary for the functioning of enzymes and other processes.

Bacterial growth is also often sensitive to temperature. Depending on the species, bacteria exhibit a usually limited range in temperatures in which they can growth and reproduce. For example, bacteria known as mesophiles prefer temperatures from 20°�° C (68°�° F). Outside this range, growth and even survival is limited. Other factors, which vary depending on species, required for growth include oxygen level, pH, osmotic pressure, light and moisture.

The events of growth and division that are apparent from measurement of the numbers of living bacteria are the manifestation of a number of molecular events. At the level of the individual bacterium, the process of growth and replication is known as binary division. Binary division occurs in stages. First, the parent bacterium grows and becomes larger. Next, the genetic material inside the bacterium uncoils from the normal helical configuration and replicates. The two copies of the genetic material migrate to either end of the bacterium. Then a cross-wall known as a septum is initiated almost precisely at the middle of the bacterium. The septum grows inward as a ring from the inner surface of the membrane. When the septum is complete, an inner wall has been formed, which divides the parent bacterium into two so-called daughter bacteria. This whole process represents the generation time.


Anaerobic Bacteria Examples

An easy way to distinguish one species from the other is by observing their body shape. Bacteria can be found in shapes like round, spiral, oblong etc.

Bacilli

This species is normally found in the intestinal tract of humans. There are friendly as well as harmful bacteria like Lactobacilli, Bifidobacterium and Escherichia.

Salmonella and Shingella are known causes of food poisoning and food-borne diarrhea.

Another type of bacillus is E. coli. E. coli is a facultative anaerobe (which can survive without oxygen) that is named after its discoverer Theodor Escherich. It is a common bacterium and is found in the intestinal tract of human beings, birds and other mammals. E. coli can cause acute respiratory problems, diarrhea and urinary tract infections.

Among the several types of E. coli that are found, E. coli 0157:H7 is the most toxic. The existence of this bacteria was discovered in the 1980s, when there was an outbreak of food poisoning caused due to undercooked beef in contaminated hamburgers. Shiga toxin, produced by this bacterium is one of the most potent poisons and is lethal for human beings.

Bacteroides

These are aerotolerant and non-spore forming anaerobes. Bacteroides can infect several parts of the human body like the peritoneal cavity and the female urogenital tract. Soft tissue damage can also occur due to endotoxins, produced by the bacteria. Prolonged exposure to these enzymes results in permanent destruction of body tissues. Since bacteroides are resistant to antibiotics, it is very difficult to treat infections. But there are a few species of bacteroides, which are beneficial to human beings.

Clostridium Genus

They are obligate anaerobes, which appear as rod shaped when observed under the microscope. There are three types found under Clostridium genus.

  • C. botulinum, is normally found in improperly handled meats and produces the deadliest toxin in the world, botulinum.

  • C. tetani, is found as parasites in the gastrointestinal tract of animals and as spores in the soil. It produces a toxin called tetanospasmin, which causes tetanus, a disease which creates painful muscle spasms leading to respiratory failure.

  • C. perfringens, earlier known as C. welchii, can be found in dead, decaying vegetation, marine sediments and in the human intestinal tract. It causes infections like food poisoning, gas gangrene and tissue necrosis.

Staphylococcus genus

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Being facultative anaerobes, this genus is found on the mucous membranes or human skin. Normally harmless in nature, this microorganism is passed from one individual to another through casual physical contact. These protozoa have resistance against antibiotics and produce a number of infections in the body. Infections can be categorized as – skin infections and invasive infections.

One type of Staphylococcus aureus causes a range of infections from ordinary skin infections like boils, acne, impetigo, etc. to life-threatening invasive infections like meningitis, endocarditis (an infection of the heart’s lining), pneumonia, blood poisoning, and toxic shock syndrome (TSS) etc.

Anaerobic Bacterial Infection

A sudden increase in the number of bacteria can trigger a series of infections. It is necessary to recognize the symptoms exhibited, in the initial stages, and treat them to avoid further complications. A few of them have been listed below:

  • Sexually transmitted diseases (STDs) like syphilis, gonorrhea and chlamydia
  • Streptococcus pneumoniae (pneumococcus) causes sinus infections in human beings.
  • Many other anaerobes also cause cold, fever, ear infections etc.
  • Lung infections like chronic bronchitis

Bacterial Infection Treatment

Infectious diseases caused by this genus can be treated as follows:

  • One must maintain proper hygiene in order to avoid infections. Keeping surroundings clean and dirt free is essential.
  • Eating a healthy diet and increasing fluid intake will help ward off infections.
  • Taking antibiotics like penicillin, quinolone, aminoglycosides, tetracyclines, etc. will help cure infections.
  • If you are affected by any of the above infections, it is advisable to seek medical help immediately.

Though some anaerobic bacteria are harmful, they play an important role of maintaining balance in the environment by keeping it clean. Taking necessary precautions will help prevent the occurrence of any infection.

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