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Can fermentation and aerobic respiration occur at the same time?

Can fermentation and aerobic respiration occur at the same time?


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In muscle cells during exercise, does lactic acid fermentation and aeorobic respiration occur at the same time, and does this mean the cell makes more or less ATP during this time?

The cell can't completely lack oxygen, which means that some of pyruvate will move into the mitochondrion, however, lactic acid is also produced which means that anaerobic respiration occurs. Is this reasoning correct or is there some other mechanism that I am skipping that doesn't allow these processes to simultaneously occur?

Also, the NAD+ used for glycolysis is regenerated and net 2 ATP is releaseD in glycolysis, but the NADH doesn't move into the mitochondrion, so if pyruvate moves into the mitochondrion at the end of glycolysis, then there will be less NADH in the mitochondrion, which makes sense because there is too little oxygen and too many electrons in the ETC.


Can fermentation and aerobic respiration occur at the same time? - Biology

EXAMPLES OF GOOD AND BAD ANSWERS


A Short Answer Question

Your roommate decides to try his hand at home-brewing beer. He adds baker's yeast, malt, hops, and sugar into a gallon of water. He then puts this mix into a gallon bucket without a lid. You wait to see if he will cover the bucket, but he leaves the mixture exposed to the air. "You know you'll never get any alcohol or carbonation produced like that," you advise your roommate.

Yeast are facultatively anaerobic which means that they perform fermentation only under anaerobic conditions. In the presence of O2, the yeast will perform aerobic metabolism. The roommate has left the bucket open to the air, so the yeast will have access to a continuous supply of O2. With O2, the yeast will replenish their NAD + through electron transport in the mitochondria. Alcohol and CO2 (which produces carbonation) are produced by the fermentation pathway which occurs significantly only in the absence of O2.

  • Irrelevant information is left out.
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Yeast are facultatively anaerobic which means that they perform fermentation only under anaerobic conditions . In the presence of O2, the yeast will perform aerobic metabolism . The roommate has left the bucket open to the air, so the yeast will have access to a continuous supply of O2. With O2, the yeast will replenish their NAD + through electron transport in the mitochondria . Alcohol and CO2 (which produces carbonation) are produced by the fermentation pathway which occurs significantly only in the absence of O2.

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  • Use of slang.
  • Teleology and anthropomorphism.

Yeast are facultatively anaerobic which means that they perform fermentation only under anaerobic conditions. In the presence of O2, the yeast will perform aerobic metabolism. The roommate has left the bucket open to the air, so the yeast will have access to a continuous supply of O2. With O2, the yeast will replenish their NAD + through electron transport in the mitochondria. Alcohol and CO2 (which produces carbonation) are produced by the fermentation pathway which occurs significantly only in the absence of O2.

Yeast are facultatively anaerobic which means that they perform fermentation only under anaerobic conditions. In the presence of O2, the yeast will perform aerobic metabolism. The roommate has left the bucket open to the air, so the yeast will have access to a continuous supply of O2. With O2, the yeast will replenish their NAD + through electron transport in the mitochondria. Alcohol and CO2 (which produces carbonation) are produced by the fermentation pathway which occurs significantly only in the absence of O2.

It addresses a different question.

Yeast are facultatively anaerobic which means that they perform fermentation only under anaerobic conditions. In the presence of O2, the yeast will perform aerobic metabolism. The roommate has left the bucket open to the air, so the yeast will have access to a continuous supply of O2. With O2, the yeast will replenish their NAD + through electron transport in the mitochondria. Yeast perform the following reaction only in anaerobic conditions.

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Engineering Fundamentals of Biotechnology

2.40.2.2.2 Oxygen uptake rate

In an aerobic fermentation , 192 g of oxygen is needed to completely oxidize 1 mol of glucose (180 g) into CO2 and H2O. This means that 1.066 g-O2 is consumed per gram of glucose:

Substrate glucose can be used for biomass formation, maintenance energy, and product formation. When biomass is formed from glucose, net oxygen requirement is decided by the difference of required oxygen for burning biomass (C5H7O2N, MW = 113, 1.42 g-O2 g-biomass −1 ) and glucose. For instance, biomass yield from glucose is 50%, 2.13 g-O2 is needed but 1.42 g-O2 is saved in the reduced form in the biomass. Thus, in this process, the next oxygen requirement is not 2.13 g but 0.71 g.

When NH3, a protein breakdown product, is present in a wastewater, the things become more complicated. NH 4 + is oxidized to NO 2 − and NO 3 − by Nitrosomonas and Nitrobacter and reduced further to N2 from NO 2 − by denitrifying bacteria. The nitrification reaction needs oxygen but the latter denitrification releases oxygen [105] . Thus, it is necessary to follow oxidation or reduction of any substrates or production theoretically and to confirm the net OUR of any reactions involving microbes experimentally.

With 8 mg of dissolved oxygen in water, 21% O2 in the air is in equilibrium, its ratio per liter is as follows:

If a glucose concentration in fermentation solution is around 80 g l −1 , the ratio of glucose to oxygen in a solution becomes 10 000. Thus, the rate-limiting step in aerobic fermentation will be a supply of oxygen from air rather than feeding of glucose.

The OUR of yeasts by respiration of glucose or aliphatic hydrocarbons will be following stoichiometric equations:

Table 3 shows specific OURs of various microorganisms, reported maximum cell densities and calculated volumetric OUR. Two unusually high cell densities of 600 and 550 g l −1 come from experimental measurements of the cells packed in the space volume among the two hollow fibers: one porous polypropylene (PP) supplies liquid medium and the other silicone tube supplies pure oxygen of dual hollow-fiber bioreactors (DHFBRs).

Table 3 . Maximum cell densities in immobilized and suspension cultures [16]

Microbial, plant, animal cellsSpecific OUR (mM O2 g −1 h −1 )Cell densityVolumetric OUR (mM O2 g −1 h −1 )Remark
Escherichia coli ( Chang et al., 1986 ) [17] [18, 33, 32, 106] 10.8600 g l −1 6480 (5–90)Facultative bacteria
Saccharomyces cerevisiae8.0210 g l −1 1680Facultative yeast
Aerobic cells6.6724.7 g l −1 165.2Suspension culture
Aspergillus niger ( Chang et al., 1986 ) [17] [18, 33, 32, 106] 3.0100 g l −1 300Fungi
Nocardia mediterranei ( Chang et al., 1986 ) [17] [18, 33, 32, 106] 3.0550 g l −1 1650Bacteria (Streptomyces)
CHO cells [39] 3.2 × 10 10 mM cell −1 h −1 5 × 10 8 cells ml −1 160Animal cells
Catharanthus roseus [116] 0.235 g l −1 7.0Plant cells

These cells of high cell density may not be metabolically as active as those of live cells or cells in suspension.


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Factors Affecting the Rate of Aerobic Respiration: 10 Factors

The following points highlight the ten factors affecting the the rate of aerobic respiration. The factors are: (1) Protoplasmic Conditions (2) Temperature (3) Supply of Oxidisable Food (4) Oxygen Concentration of the Atmosphere (5) Carbon Dioxide Concentration of the Atmosphere (6) Supply of Water (7) Light (8) Inorganic Salts (9) Injury and the Effects of Mechanical Stimulation and (10) Effect of Various Chemical Substances.

Factor # 1. Protoplasmic Conditions:

Young actively growing meristematic tissues have always higher rates of respiration than older and more mature parts. The proportion of protoplasm, both relative and absolute, is always greater in the young cells compared to maturer, vacuolated cells. There seems to be a direct relationship between the amount of protoplasm and the respiration rates—the greater the protoplasm, the higher is the respiration rate. Hydration of protoplasm and the quantity of respiratory enzymes of the mitochondria (all the respiratory Krebs cycle enzymes are known to occur, in mito­chondria) are important protoplasmic factors which contribute to the effects obtained.

Factor # 2. Temperature:

Within certain limits (0-45°C.) increase in temperature leads to an increase in the initial respiration rate. Above 30°C., the respiration rate slows down and the decrease above the optimum may possibly be due to the progressive inactivation of respiratory enzymes. Other causes which might contribute to this lower­ing of respiration rate may include: (a) O2 may not get access to the cell fast enough, (b) CO2 may accumulate in toxic concentration, (c) the supply of oxidisable respiratory substrate may be inadequate to keep pace with the high rates of respiration, etc. At 0°C., respiration rate greatly diminishes and soon becomes imperceptible though there are some records of measurable respiration even at — 20°C. The decline of respiration at temperatures below zero can be attributed to the formation of ice and consequent dehydration of protoplasm.

The temperature coefficient (Q10) of respiration within the temperature range of 0-35°C appears to be about 2.0 to 2.5.

Factor # 3. Supply of Oxidisable Food:

Increase in soluble food content readily available for utilisation as respiratory substrate, generally leads to an increase in the rate of respira­tion up to a certain point when some other factor becomes limiting.

Factor # 4. Oxygen Concentration of the Atmosphere:

In considering first the effect of oxygen concentration in gas mixtures at N.T.P., the whole range of oxygen concentrations falls in two clear cut divisions, a lower range, in which anaerobic respiration occurs and an upper range, in which the entire volume of CO2 evolved results from aerobic respiration.

The lower range extends from zero to the extinction point (generally about 4-5% oxygen) which, as we know, is that concentration of oxygen just high enough to completely suppress the anaerobic component. The upper range extends from the extinction point to 100% Oxygen.

In general, the rate of respiration is decreased by oxygen concentration smaller than that of air (21%) which drops off very sharply at concentration of oxygen less than 5-10%.

The oxygen poisoning, i.e., the significant fall in respiration rate, was observed in many tissues in pure O2, even at N.T.P. This inhibiting effect was also observed with green peas when they were exposed to pure oxygen exerting a pressure of 5 atm.— the respiration rate fell rapidly. The oxygen poisoning effect was reversible, if the exposure to high oxygen pressure was not too prolonged.

The relation of oxygen concentration to respiration has a particularly important implication in the growth of the roots. Roots must respire vigorously if they are to grow and take up minerals from the soil by active absorption. The vigorous root respiration is only possible if the space surrounding the roots and root hairs has ample supply of oxygen. When the roots are poorly aerated as in water-logged or heavy soils, growth of the plant may be significantly restricted.

Factor # 5. Carbon Dioxide Concentration of the Atmosphere:

In general, the higher the con­centration of CO2 of the atmosphere, the lower is the rate of respiration. This fact is made use of in storage of fruit. Air containing 10% CO2 (in atmosphere it is only 0.03%) retards respiratory breakdown and therefore reduces sugar consumption and thus prolongs the life of the fruit.

The oxygen content of the air, however, must be maintained as high as normal to prevent anaerobic respiration. In some plant tissues, however, respiration rate actually increases when exposed to relatively high concentration of CO2. It is thought now that the effect of CO2-concentration on the respiration rate is influenced not only by its concentration in the medium but also depends upon the kind of tissue and the period of exposure.

Factor # 6. Supply of Water:

Increase in the percentage of moisture leads to a general increase in respiration rate. This increase is slow at first but very rapid later. This is very clearly seen in the tissues of many xerophytes. As the water content of such plants is increased, often there is no great immediate effect upon the rate of respira­tion, until a certain water content (which varies according to the tissue) is attained after which respiration rate shows rapid increase. On the other hand, minor variations in water content of well-hydrated plant cells do not appear to have very great influ­ence upon the rate of respiration.

Factor # 7. Light:

As far as we know light has no direct effect on respiration except in bringing about an increase in temperature which certainly, as we know well, influences respiration. The indirect effect of light on respiration is, of course, immense because only in light the primary respiratory substrates are synthesised.

That light has no direct effect on respiratory activity has been shown in some mutant strains of Chlorella which, although green, cannot photosynthesise. These algae, however, grow and respire when supplied with a suitable source of carbon. The respira­tion rate of these forms was entirely unaffected by illumination. With blue-green alga, Anabaena, however, respiration rate was found to depend on light, and the effect was also influenced by O2-concentration.

The term photorespiration has attracted a lot of attention during the last few years. It is used to indicate increased respiratory activity in light, regardless of the pathways of respiration, by which CO2 is released and oxygen consumed.

Some plants, e.g., tobacco, evolve CO2 when brightly illuminated in CO2-free air whereas others, e.g., maize, do not. This light respiration is stimulated by high oxygen concentration of the medium. The different responses by plants to temperature also suggest that this photorespiration is different from normal mitochondrial respiration. There are strong evidences also for progressive increase in photorespiration with increa­sing light intensities.

In tropical HSK-plants (e.g., sugar cane leaves), photorespiration is very difficult to detect as these species are extremely efficient in photosynthesis. Compared to these plants, the temperate species, which fix CO2 primarily by following the classical cycle, have very high respiratory rates.

Factor # 8. Inorganic Salts:

The chlorides of alkali cations of Na and K, as also the divalent cations of Li, Ca and Mg, generally increase the rate of respiration as measured by the amount of CO2 evolved, although there is considerable difference in the effects of monovalent and divalent cations. With monovalent chlorides of K and Na, the high respiration rates may be maintained for some 7—10 days whereas with divalent chlorides of Li, Ca and Mg, the increased rate observed generally falls off after about a day. Similar results are obtained with NH4Cl but in all cases this effect seems to be transitory.

The transitory increased respiration rates on addition of chlorides, particularly of potassium and sodium may be calculated as total respiration in salts minus the ground- respiration (ordinary respiration), unconnected with salt uptake. The ground respiration is essentially cyanide resistant whereas the extra respiration due to salt addition was found to be cyanide sensitive—10 -4 M cyanide completely abolished the increased respiration.

This enhanced respiration has been termed salt respiration or anion respiration which, according to Lundegardh, was directly related to the total amount of anion absorbed by plant cells rather than to the absorption of cations.

Factor # 9. Injury and the Effects of Mechanical Stimulation:

Wounding or injury almost invariably results in an increase in the rate of respiration. Broken and shrivelled seeds and kernels have always higher respiration intensities than clean, intact seeds of the same type and moisture content. It is possible that the increased respiration rate associated with injury may partly he correlated to an increase in the sugar content, actually observed in the cells close to the injury, which-may be as high as 70% com­pared to intact parts. This increased rate of respiration following injury is not, however, maintained for more than 48—72 hours.

A considerably higher respiration rate is observed when leaves are first touched or handled and this effect could also be produced by stroking or bending the leaf by a mechanical arrangement. The possibility that mechanical stimulation actually affects the oxidation process itself is supported by the observation that mechanical stimulation has no effect on respiration rate in an atmosphere of only nitrogen.

The effect of two successive stimulation with a short interval between them is interesting. Both the first and second stimulations increase the respiration rate but although the respiration rate reached as a result of second stimulation may be actually higher than the peak reached by the first stimulation, the net increase in respiration rate after the second stimulation is always less than that resulting from the first stimulation, if the time between the two stimulations is less than three days. It is evident that the sensitivity of a tissue to respira­tory mechanical stimulation is lowered by a previous stimulation.

Factor # 10. Effect of Various Chemical Substances:

Various chemical substances such as chloro­form, ether, acetone, morphine, etc., temporarily increase respiration rates if given in small doses but if the dose is large, a rapid decrease in respiration rate is observed.


4.4 Fermentation

In aerobic respiration, the final electron acceptor is an oxygen molecule, O2. If aerobic respiration occurs, then ATP will be produced using the energy of the high-energy electrons carried by NADH or FADH2 to the electron transport chain. If aerobic respiration does not occur, NADH must be reoxidized to NAD + for reuse as an electron carrier for glycolysis to continue. How is this done? Some living systems use an organic molecule as the final electron acceptor. Processes that use an organic molecule to regenerate NAD + from NADH are collectively referred to as fermentation . In contrast, some living systems use an inorganic molecule (other than oxygen) as a final electron acceptor to regenerate NAD + both methods are anaerobic (do not require oxygen) to achieve NAD + regeneration and enable organisms to convert energy for their use in the absence of oxygen.

Lactic Acid Fermentation

The fermentation method used by animals and some bacteria like those in yogurt is lactic acid fermentation (Figure 4.16). This occurs routinely in mammalian red blood cells and in skeletal muscle that has insufficient oxygen supply to allow aerobic respiration to continue (that is, in muscles used to the point of fatigue). In muscles, lactic acid produced by fermentation must be removed by the blood circulation and brought to the liver for further metabolism. The chemical reaction of lactic acid fermentation is the following:

The enzyme that catalyzes this reaction is lactate dehydrogenase. The reaction can proceed in either direction, but the left-to-right reaction is inhibited by acidic conditions. This lactic acid build-up causes muscle stiffness and fatigue. Once the lactic acid has been removed from the muscle and is circulated to the liver, it can be converted back to pyruvic acid and further catabolized for energy.

Visual Connection

Tremetol, a metabolic poison found in white snake root plant, prevents the metabolism of lactate. When cows eat this plant, Tremetol is concentrated in the milk. 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?

Alcohol Fermentation

Another familiar fermentation process is alcohol fermentation (Figure 4.17), which produces ethanol, an alcohol. The alcohol fermentation reaction is the following:

In the first reaction, a carboxyl group is removed from pyruvic acid, releasing carbon dioxide as a gas. The loss of carbon dioxide reduces the molecule by one carbon atom, making acetaldehyde. The second reaction removes an electron from NADH, forming NAD + and producing ethanol from the acetaldehyde, which accepts the electron. The fermentation of pyruvic acid by yeast produces the ethanol found in alcoholic beverages (Figure 4.18). If the carbon dioxide produced by the reaction is not vented from the fermentation chamber, for example in beer and sparkling wines, it remains dissolved in the medium until the pressure is released. Ethanol above 12 percent is toxic to yeast, so natural levels of alcohol in wine occur at a maximum of 12 percent.

Anaerobic Cellular Respiration

Certain prokaryotes, including some species of bacteria and Archaea, use anaerobic respiration. For example, the group of Archaea called methanogens reduces carbon dioxide to methane to oxidize NADH. These microorganisms are found in soil and in the digestive tracts of ruminants, such as cows and sheep. Similarly, sulfate-reducing bacteria and Archaea, most of which are anaerobic (Figure 4.19), reduce sulfate to hydrogen sulfide to regenerate NAD + from NADH.

Concepts in Action

Watch this video to see anaerobic cellular respiration in action.

Other fermentation methods occur in bacteria. Many prokaryotes are facultatively anaerobic. This means that they can switch between aerobic respiration and fermentation, depending on the availability of oxygen. Certain prokaryotes, like Clostridia bacteria, are obligate anaerobes. Obligate anaerobes live and grow in the absence of molecular oxygen. Oxygen is a poison to these microorganisms and kills them upon exposure. It should be noted that all forms of fermentation, except lactic acid fermentation, produce gas. The production of particular types of gas is used as an indicator of the fermentation of specific carbohydrates, which plays a role in the laboratory identification of the bacteria. The various methods of fermentation are used by different organisms to ensure an adequate supply of NAD + for the sixth step in glycolysis. Without these pathways, that step would not occur, and no ATP would be harvested from the breakdown of glucose.

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    How do fermentation and anaerobic respiration differ?

    Fermentation and anaerobic respiration differ because although they both start with glycolysis, fermentation does not stop with the product of glycolysis, but instead creates pyruvate and continues on the same path as aerobic respiration.

    Explanation:

    Adenosine Triphosphate (ATP) is the chemical form of energy . There are many different mechanisms that can convert the original energy source into ATP. The most efficient way is through aerobic respiration, which requires oxygen. This method will give the most ATP per input energy source. However, if no oxygen is available, the organism must still convert the energy using other means. Processes that happen without oxygen are called anaerobic. Fermentation is a common way for living things to continue making ATP without oxygen.

    UNDERSTANDING FERMANTATION
    Aerobic respiration begins with a process called glycolysis. In glycolysis, a carbohydrate (such as glucose) gets broken down and, after losing some electrons, forms a molecule called pyruvate. If there is a sufficient supply of oxygen, or sometimes other types of electron acceptors, the pyruvate then goes on to the next part of aerobic respiration. The process of glycolysis will make a net gain of 2 ATP.

    Fermentation is essentially the same process. The carbohydrate gets broken down, but instead of making pyruvate, the final product is a different molecule depending on the type of fermentation. (due to lack of oxygen) In Humans Instead of pyruvate, lactic acid is formed. Long distance runners are familiar with lactic acid. It can build up in the muscles and cause cramping.. Since fermentation does not use the electron transport chain, it is not considered a type of respiration.

    UNDERSTANDING ANAEROBIC RESPIRATION
    Anaerobic respiration begins the same way as aerobic respiration and fermentation. The first step is still glycolysis and it still creates 2 ATP from one carbohydrate molecule. However, instead of just ending with the product of glycolysis it will create pyruvate and then continue on the same path as aerobic respiration.

    After making a molecule called acetyl coenzyme A, it continues on into the citric acid cycle. More electron carriers are made and then everything ends up at the electron transport chain. The electron carriers deposit the electrons at the beginning of the chain and then, through a process called chemiosmosis, . If the final electron acceptor is oxygen, the process is considered aerobic respiration. and like many types of bacteria and other microorganisms, can use different final electron acceptors. That is called anaerobic respiration.

    EVOLUTION
    Scientists believe that fermentation and anaerobic respiration are more ancient processes than aerobic respiration. Lack of oxygen in the early Earth's atmosphere made aerobic respiration impossible at first.


    BIOLOGY ORDINARY LEVEL NOTES

    Respiration is necessary to supply organisms with energy which help them to maintain themselves, move, excrete, grow and reproduce. The main source of energy for organisms is the radiant energy from sunlight. During photosynthesis, green plants transofrm this energy into chemical energy which is stored in the organic foods (products of photosynthesis). This stored chemical energy is obtained by orgnisms (animals) by feeding on green plants or on other animals.

    HOW DOES THIS STORED CHEMICAL ENERGY RELEASED FROM THE FOOD?
    since the energy is locked up in the organic food molecules, an organism will have to oxidise them. This process of oxidation which occurs within every living cells of animals (aw well as plants) is called respiration.

    IMPORTANT:
    When students were asked about their pre-conceptions about respiration, the common misconception was that respiration is thought to be a scientific name for breathing. NO. Totally WRONG. But breathing does help indirectly by bringing in oxygen into your body.

    DEFINITION OF RESPIRATION IN GENERAL:
    Respiration is the oxidation of food substances (mainly glucose) with the release of energy in living cells.

    Note: Do Not use BURNING or BREAK DOWN. Stick to OXIDATION. and it is very important for you to remember that respiration occurs within living cells and where in the cells? The MITOCHONDRIA.

    Respiration can be of two forms: AEROBIC RESPIRATION and ANAEROBIC RESPIRATION.

    AEROBIC RESPIRATION
    Aerobic respiration is defined as the oxidation of food substances in the presence of oxygen with the release of a large amount of energy and carbon dioxide and water as the waste products.

    Aerobic respiration can be represented by the following equation.

    C 6 H 12 O 6 + 6O 2 --> 6CO 2 + 6H 2 O + Energy

    How much energy? 17.1kJ/g of glucose.

    The process shown by the above equation involves many enzyme-catalysed reactions. The enzymes for each of these enzyme-catalysed reactions are found in the mitochondria. Hence mitochondria are important in respiration. In fact it is the site where respiration occurs.

    • Synthesis of proteins from amino acids
    • Cell division and therefore growth
    • Heartbeat
    • Respiratory movements
    • Muscular contractions
    • Active transport (absorption of glucose and amino acids by the villi in the small intestine is by active tranport. So does absorption of mineral salts by the root hair cells)
    • Transmission of nerve impulses

    ANAEROBIC RESPIRATION
    Anaerobic respiration is defined as the oxidation of food substances in the absence of oxygen with the release of a small amount of energy.

    ANAEROBIC RESPIRATION IN YEAST (FERMENTATION)
    Yeast is a kind of fungi which is used in bread making. Yeast cells oxidise glucose during frementation. The products is ethanol which is an alcohol. For this reason, anaerobic respiration is yeast is also called alcoholic fermentation. Anaerobic respiration is yeast can be represented by the following equation:

    C 6 H 12 O 6 --> 2C 2 H 5 OH + 2CO 2 + Energy

    How much energy is released? 1.17kJ/g glucose.

    Less energy is released because the alcohol molecule is relatively large and still contains a conisderable amount of chemical energy. The small amount of energy released is only enough for the yeast to survive.

    (Note: the carbon dioxide produced as the waste products actually helps in raising the bread dough thus making the bread fluffy)

    (Note: wine is also made in a similar way. The glucose in grapes is oxidised in the same way)

    ANAEROBIC RESPIRATION IN MUSLCES
    Normally muscle cells respire aerobically (using oxygen gas). But in certain circumstances, where oxygen is not available for a very short period of time, these cells need to respire anaerobically (without oxygen gas). This usually occurs during a vigorous or strenous exercise such as sprinting in a 100m race.


    Top 16 Experiments on Respiration in Plants (With Diagram)

    The following points highlight the top sixteen experiments on respiration in plants. Some of the experiments are: 1. Demonstration of Aerobic Respiration in Plants 2. Demonstration of Anaerobic Respiration 3. Demonstration of Alcoholic Fermentation 4. Determination of Rate of Respiration 5. Comparison of Rate of Respiration in different Plant Parts and Others.

    Experiment # 1

    Demonstration of Aerobic Respiration in Plants:

    Aerobic respiration in plants can be experimentally proved with the help of a simple apparatus like:

    (i) Respiroscope which consists essentially of a stout vertical tube which is bent into a bulb at the one end (Figure 19a), or

    (ii) With the help of a long-necked round-bottomed flask fitted with a centrally-bored cork at the mouth through which passes a glass tube (Figure 19b).

    The respiroscope or the inverted flask is fixed vertically to a stand and a few germinating gram seeds or flower petals are placed in the bulb of the respective or in the inverted flask plugged with cotton at the base the vertical tube of the respiroscope or inverted flask is dipped just below the surface of water or mercury in a beaker.

    A few caustic potash (KOH) pellets are introduced in the bent portion of the respiroscope or in the long neck of the round bottom flask and kept in position with loosely held cotton wool Care should be taken that respiratory materials and KOH pellets do not come in contact.

    Precautions should be taken that the free end of the tube does not touch the bottom of the water or mercury trough. Fittings must be air-tight to avoid any leakage.

    The apparatus is allowed to stand for a few hours when it is seen that water or mercury has risen in the vertical tube of the apparatus proving the production of partial vacuum.

    Due to respiration of germinating seeds or flower petals CO2 has been released which is at once absorbed by KOH pellets. Thus the partial vacuum produced by the absorption of O2 by the respiring material could not be filled up by the released CO2. Hence, water or mercury is drawn upward into the tube.

    Experiment # 2

    Demonstration of Anaerobic Respiration:

    A few germinating gram seeds are taken in a test tube which is completely filled with mercury and is then inverted just below the surface of mercury in a trough. It is then vertically held with a clamp and stand (Figure 20).

    Observation from time to time reveals that a gas is formed within the test tube by the displacement of mercury in the test tube. A few KOH pellets arc introduced through the open end of the test tube when mercury again rises filling the test tube.

    Here the respiration of germinating seeds takes place in complete absence of O2 supply and the gas produced is CO2 as evidenced by its absorption by KOH. This proves that anaerobic respiration has taken place.

    Experiment # 3

    Demonstration of Alcoholic Fermentation:

    A fermentation tube or Kuhne’s vessel (Figure 21) is filled with 10% sucrose solution and mixed with a small quantity of Baker’s yeast or a few millilitres of suspension of yeast cells. The open end of the apparatus is plugged with cotton wool.

    Occurrence of fermentation or anaerobic respiration and collection of CO2 gas in the back arm of the Kuhne’s tube are observed. When the cotton wool is taken off smell of alcohol may be perceived.

    In alcoholic fermentation the sugar and yeast soln. broken down to alcohol and carbon dioxide liberating certain amount of energy. The enzyme, ‘zymase complex’ present in the yeast brings about this reaction through a number of steps. Alcoholic fermentation takes place in absence of atmospheric oxygen. Hence it is an anaerobic process.

    Experiment # 4

    Determination of Rate of Respiration:

    The rate of res­piration can be measured with the help of Ganong’s respirometer (Figure 22).

    Description and Experiment:

    The apparatus consists of three Levelling tube parts:

    (i) The bulb for the res­piring material which ends in a 10% KOH win smaller bulb at the bottom. The bigger bulb is provided with a stopper having a lateral hole, through which atmospheric con­nection can be made by turning the stopper,

    (ii) A graduated manometer fitted with the bulb, and

    (iii) A levelling tube connected with the manometer tube by rubber tubing. The whole apparatus is clamped on a stand.

    Two ml of respiring material (measured by displacement of water) like germinating seeds or flower petals are placed into the bigger bulb of the respirometer. A 10% solution of KOH is taken in the manometer tube. At the beginning of the experiment the air around the material is brought to the atmospheric pressure by turning the stopper of the bulb until its hole coincides with that of the neck of the bulb.

    The levelling of the reservoir tube on the right is so adjusted that the KOH solution in the tube is at the 100 ml mark at the bottom of the manometer. Two ml of respiring material is now surrounded by 100 ml of air. The experiment is started by turning the glass stopper at the top and thus cutting off connection with the atmospheric air.

    As the respiration takes place in a closed space, the solution in the manometer tube rises up gradually. The reading should be taken up to 80 ml mark, i.e., up to 20 ml volume (since atmospheric oxygen is 20%) at an interval of 10 minutes, each time bringing the liquid in both the tubes at the same level, i.e., the liquid in the closed tube is brought under atmospheric pressure.

    Results are expressed as millilitre of CO2 evolved per minute by the given respiring material.

    The released CO2, on coming in contact with KOH solution, is absorbed by it, oxygen is consumed and as a result KOH solution rises up in the manometer tube. The rate of rise of KOH solution is taken as a measure of rate of aerobic respiration in terms of volume of O2 consumed per unit time per 2 ml of respiring material.

    One-fifth volume of atmos­pheric air is O2. Hence out of 100 ml of enclosed air within respirometer there is 20 ml of O2. Hence reading should be taken up to 20 ml rise in volume of KOH solution. After that anaerobic respiration will start.

    Experiment # 5

    Comparison of Rate of Respiration in Different Plant Parts:

    Flower buds, roots and leaves of a suitable herbaceous plant are collected 2 ml of each is measured by displacement of water and placed in the bulbs of three different Ganong’s respirometers. Rate of respiration is measured in each case following Expt. 4.

    The volume of CO2 evolved at an interval of 10 minutes is recorded in each case and rates of respiration are graphically plotted for each sample of plant material and compared.

    The rate of respiration is always higher in younger actively growing meristematic tissues than that of older and mature parts. There is a direct relationship between the amount of protoplasm and the rate of respiration the greater the protoplaim, the higher is the respiration rate.

    The hydration of protoplasm and quantity of respiratory enzymes are al­ways greater in young cells compared to mature and vacuolated cells. Hence the respiratory rate is always higher in young cells which are rich in protoplasm.

    Vigorous respiration of root takes place if the space surrounding the roots and root hairs has ample supply of oxygen. In this experi­ment the maximum rate of respiration is expected in case of flower buds than in roots and leaves.

    N.B. This experiment may also be performed with different types of seeds (starchy, proteinaceous and fatty).

    Experiment # 6

    Quantitative Estimation Of CO2 Evolved During Respiration:

    (a) By Barcroft-Warburg’s constant volume micro-respirometer:

    i. Principle and Description:

    A convenient method of measuring the respiration of tissues in minute quantities has been developed by Warburg (1926).

    The tissue whose respiration is to be measured is placed in a closed container with an attached manometer which records changes in gas pressure as a result of oxygen consumption or carbon dioxide production.

    The apparatus is shown in Figure 23. Each respirometer consists of two main parts, a glass flask f and a manometer m, separable at a ground glass joint j. The tissue is placed in the flask f. When only oxygen consumption is to be measured, Ba (OH)2 or NaOH solution is added to the well w at the centre of the flask.

    But when both oxygen consumed and carbon dioxide produced are to be measured, an HCL solution is placed in the side arm a with stoppered neck n in addition to the alkali in w.

    The manometer fluid is contained in a rubber bulb b and can be added to or withdrawn from the manometer by adjusting the screws. This enables one to return the right side of the manome­ter to the starting point during making a reading and also to read the pressure on the left arm of the apparatus at constant volume.

    A mirror behind the manometer reduces parallax in reading. The manometer is provided with a two-way tap t at the closed end. The reaction chamber is kept in a bath of constant temperature. The entire appa­ratus is shaken to facilitate gas exchange and temperature equilibrium.

    It is necessary to know the volume of the apparatus including the manometer to the manometer fluid in order to calculate gas volumes from changes in pressure. The manometer is detached and filled with clean mercury from the 15 cm mark to a marked point about 2 cm above the flask connection.

    Now mercury is poured into a beaker and the dry reaction chamber f is filled with clean mercury until it just rises to the marked point on the manometer when the manometer and flask are connected.

    This mercury is collected in a beaker and the temperature and weight of the metal are determined. The weight of the mercury in milligrams divided by its density at the observed temperature gives the volume of the apparatus in cubic millimeters.

    Brodie solution (23gm NaCL, 5gm sodium cholate, 500 ml water, 5 drops conc. thymol in alcohol, as a preservative, and a few crystals of neutral red to colour) is used in the manometer to increase the sensitivity of readings, and to avoid sticking and other difficulties. This solution has a density of 1.0336 and gives a manometric pressure of one atmosphere (760 mm Hg) at 10,000 mm.

    If in addition to these two values, the volume of the apparatus and the normal barometric height of the manometer fluid, we know the temperature, the volume of the material whose respiration is being measured, the volume of fluids (water, NaOH, etc.) added to the reaction chamber, and the solubility of the gas being measured in these contained liquids, the change in volume of the contained gases in cubic millimeters under standard conditions can be calculated with the equation

    where X is the volume of gas absorbed (- X) or evolved (+ X) in cubic millimeters (cu. mm) under standard temperature and pressure h is the manometer reading in millimeters (reading of left arm minus right) Vg is the free volume of gas in flask and manometer to manometer fluid (total volume of apparatus less volume of sample, liquids, etc., added to reaction chamber) T is the absolute temperature of the water around the reaction flask if is the volume of all fluids in which the measured gas might dis­solve (ordinarily not including volume of solid samples) a is the Bunsen coefficient of the solubility of the gas being measured in the contained fluids at the temperature T (see below) it is to be noted that Vf x a gives the volume of dissolved gas and that this is added to the free gas (Vg) to give the total volume Po is the normal pressure in terms of the manometer fluid (for Brodie solution 10,000 mm).

    In its simplest terms the equation states that the change in gas volume during the experiment is equal to the fractional change in pressure h/Po times the total volume of the gas Vg, with corrections for temperature and the solubility of the gas in the fluids present.

    The above equation assumes that the barometric pressure, the temperature, and the vapour pressure of the contained liquids remain constant during the experiment and therefore cancel out.

    In practice, the great sensitivity of the manometer makes it necessary to set up an apparatus with the liquids, but without a sample, and to correct the experimental readings by the changes in the manometer of this blank apparatus, which are due to temperature or to barometric variations during the course of the experiment.

    This method is most suitable for measuring respiration of minute quantity of respiring material. The reservoir b of the manometer is filled with Brodie solution. 1 ml of the respiring material is placed in the outer part of the flask f 0.2 to 0.4 ml of CO2 free 2N KOH solution is taken in the central cup w and 0.3 to 0.6 ml 2.5 N HCL in the side arm a.

    The manometer connections are greased lightly but uniformly. The flask is secured in place with springs or rubber bands keeping the stopcock t open. The temperature of the water bath is kept constant at 30°C.

    If only O2 consumption is to be measured, the HGL is omitted from the side arm and one or more samples are set up as desired in different flasks. If both O2 and CO2 are to be measured to obtain RQ, the HCL is included in the side arm and all experiments are set up in duplicate. In either case a flask is set up with a sample but with KOH and other fluid to serve as thermo-barometer.

    The stopcock t is left open. The assembled manometers and flasks are set in the water bath and shaken for 15 minutes to attain temperature equilibrium. Now with screw is the right arm of the manometers is ad­justed to 250 point for which they are calibrated (150 to 250 mm), the stopcocks are closed and the time is recorded as the beginning of the experi­ment.

    When CO2 production is to be measured, the duplicate flask for each sample is quickly removed, a finger is held tightly over the open end of the manometer to prevent the manometer fluid being blown out or sucked into the flask, the flask is tipped to mix the KOH and HCL solutions thoroughly from the cup w and arm a.

    These flasks are returned to the bath the manometer is carefully released, shaken for 5 to 8 minutes and manometer reading is recorded. This is corrected by changes in the thermo-barometer, as CO2 present or produced before experimental time.

    The remaining flasks are shaken for 100 to 130 oscillations per minute for one hour or more. Intermediate readings for O2 consumption may be made as desired by stopping the shaker and adjusting the right arms of the manometers to the original point and recording the manometer readings including that of the thermo-barometer.

    At the end of the experiment, the total O2 consumption is recorded and absorbed CO2 is liberated by the method used for the control samples taking care to protect the manometer fluid against changes in pressure and to bring the gases at water bath temperature with vigorous shaking before reading the CO2 pressure.

    The change in pressure upon the mixing of KOH and HCL gives the ‘h’ reading for CO2. The right side of the mano­meter arm is always returned to its original setting before taking a reading since all of the calculations are based upon a constant volume of gas within the apparatus. The thermo-barometer pressure is always recorded along with each reading.

    The proper terms are substituted in the equation

    The gas absorbed or evolved is calculated in cu. mm, or in ml per gram of dry tissue per hour. The value Vg varies with the flask and with the volume of added samples or other fluids.

    The volume of bacterial cultures and of KOH and HCL solution is obtained by pipetting the volume of seeds, tissues, etc., by displacement. Vf is usually the volume of sample and other fluids for bacterial cultures but does not include the volume of seed tissue.

    In experiments in which a constant volume of sample is run at constant temperature, the value of the quantity within the brackets remains constant and can be assigned a value K (flask constant) so that the equation becomes X = AK, in which K is calculated for each flask at each temperature.

    The result may be expressed in mg by multiplying the volume of CO2 in ml by the density of CO2 at i particular temperature and pressure.

    The effects of temperature or other factors upon the respiration and RQ of seeds and plant tissues may be measured by this apparatus.

    (b) By Pettenkoffer’s gas stream method of CO2 estimation Principle and description of apparatus:

    The principle of this method is that CO2 liberated by respiratory tissue is removed from its chamber along with CO2 free gas stream and absorbed in baryta (Barium hydroxide solution) taken in Pettenkoffer’s tube to form barium carbonate.

    This is then titrated by a standard acid (HCL) to know its CO2 content. CO2 free air is drawn through a system as shown in Figure 24.

    At the extreme left there is a soda lime tower containing soda lime for absorbing CO2 of the air entering through its opening at the mouth. The end of the tower is fitted with a tube through which CO2 free air passes into a U-tube containing KOH solution (30%).

    The U-tube in turn is connected with another tower containing lime water or B(OH)2 solution which is connected to the respiration chamber by means of a tube. The respiration chamber is again connected to one end of the horizontally placed Pettenkoffer’s tube containing 50 ml of standard N/10 B(OH)2 solution (8-567gm/litre).

    The other end of this tube is connected to an aspirator or suction pump. Thus on applying suction at the extreme right, air current enters the system of towers and tubes through the inlet at the top of the soda lime tower.

    A mercury trough may be introduced in between Pettenkoffer’s tubes and the aspirator to regulate air flow. All connections should be made air-tight.

    At the beginning of experiment, weighed amount of plant tissue is taken in the respiration chamber. All the towers and Petten­koffer’s tubes are connected as described and made air-tight. The inlets of KOH tube and baryta tower must be dipped into the solution but the exit tubes should remain well above the surface of the solution.

    Now the aspirator or suction pump is started. Air is sucked out through the end of the Pettenkoffer’s tube at the aspirator end causing the air to bubble into the solution of baryta contained in the Pettenkoffer’s tube through the respiration chamber and other towers successively.

    Thus air first comes through the soda lime tower and then through KOH tube and baryta tower, thus becoming completely free of CO2. This CO2 free air containing O2 comes in contact with the plant tissue in the respiration chamber and aerobic respiration takes place as a result of which CO2 is evolved.

    This CO2 produced by plant tissue during aerobic respiration then passes through the standard baryta solution of Pettenkoffer’s tube and is completely absorbed by it forming BaCO3.

    After allowing respiration to occur for a particular time Pettenkoffer’s tube is taken out and the excess baryta is titrated against Standard N/10 HCl using phenolphthalein as an indicator to estimate the quantity of CO2 from the following relations.

    The baryta and the BaCO3 solution of the Pettenkoffer’s tube is quantitatively transferred in a flask, a drop or two of phenolph­thalein solution is added and titrated against N/10 HCL until the pink colour is just discharged.

    This gives the volume of residual Ba (HO)2 (not utilised by the CO2 formed). This titration value is subtracted from the titration value of fresh 50 ml sample of Ba (OH)2 solution and weight of CO2 liberated in respiration is calculated from the following equation:

    Where Vis the difference between blank and experimental titration values in millilitres, N is the normality of acid used in titration and 22 is the equi­valent weight of CO2 in BaCO3. Since molecular weight of CO2 is 44 and it is absorbed by Ba (OH)2 as H2CO3 the equivalent weight is to be calculated by dividing the molecular weight by 2.

    This method sometimes called the “gas-stream method”, has the advantage of accuracy and convenience and of permitting a study of the cell material for an indefinite period under constant conditions.

    Here the CO2 yield is obtained in milligrams the result may be divided by thou­sand to give grams or divided by 1-977 (density of CO2) to change to ml of CO2 at 0°C and 760 mm. Hg. It is to be noted that CO2 is calculated directly rather than as the carbonic acid which is actually measured.

    Experiment # 7

    Determination of Respiratory Quotients (RQ) by Ganong’s Respirometer:

    From this experiment, volume of CO2 evolved and O2 consumed during aerobic respiration can be directly obtained and RQ,, i.e., (volume of CO2/volume of O2) can be calculated.

    The experiment can be conveniently performed by Ganong’s respirometer described in Expt. 4. The manometer tube and reservoir are filled with brine solution (saturated NaCl solution) and by adjusting the reservoir, the surface of the brine solution is brought to 100 ml mark of manometer.

    The weighed equal quantities (2 ml) of tissues whose RQ, is to be determined are taken in the bulbs of two similar types of Ganong’s respirometer. In the bulb of one respirometer 1 ml of 40% KOH solution contained in a small vial is kept along with the tissue.

    Total volume of tissue plus KOH solution plus vial should be noted (let it be k ml). The other respirometer contains only tissue in the bulb and its volume is noted (let it be t ml). Now the air within the bulb is brought to atmospheric pressure by turning the stopper.

    The level of brine solution is brought to 100 ml mark at the bottom of manometer. This is done in both the respirometers and the connection with the outside air is cut off by turning the stoppers. Now the bulb and the manometer tube up to the level of brine solution contain a definite volume of air having 20% 02 and 0.03% of CO2 on an average.

    The setup is placed in dark or covered with black paper for a certain period of time (say two hours) and any change in the reading of brine level is noted. The volume of air in case of the respirometer containing plant tissue only is 100—t and this multiplied by 20/100 gives the volume of O2 in this air.

    Similarly the volume of air in case of the respirometer containing KOH vial and the plant tissue is 100 —k and this multiplied by 20/100 gives the O2 in this closed atmosphere.

    During the time for which the setup is kept, respiration occurs in the tissues of both the respirometers by absorbing O2 and giving out CO2. In the respirometer containing KOH via), CO2 given out is absorbed by KOH solution but this does not happen in the respirometer without KOH3. Therefore, the brine level rises in the respirometer with KOH and either rises or falls or remains stationary in the respirometer without KOH.

    In the respirometer containing KOH, CO2 is absorbed reducing the pressure in the manometer tube and brine level rises because O2 has been used up by the tissue. The more O2 is used up, the more raises the brine level.

    Now by adjusting the reservoir the brine level is brought to the same level in both the arms, thus bringing the volume of air in the manometer tube in atmospheric pressure. At this stage the brine level in manometer tube is noted and the difference between the final arid initial readings gives the volume of O2 used by the tissue (let it be x ml).

    Now in a respirometer without KOH, O2 is also used up giving out CO2. But in this case CO2 is not absorbed since KOH is not present. If CO2given out is less than O2 used (when fat is a substrate) the brine level will rise in the manometer tube.

    If CO2 given out is more than O2 used (when acid is a substrate) the brine level will fall down in the manometer tube and when CO2 given out is equal to O2 used up (when carbohydrate is a substrate), the level of brine remains stationary. Before taking readings the brine level is adjusted at the same level in both the arms.

    The difference between the final and initial readings gives the net gas exchange which has taken place (let it be y ml). If the rise of brine level upward is considered as negative change and fall of brine level downward is considered as positive change, the volume of CO2 evolved will be (x – y) ml when brine level rises up and (x + y) ml when brine level falls down. It may also remain stationary when RQ, is unity.

    Unity when the brine level in the respirometer without KOH remains stationary indicating that the respiring substrate is carbohydrate.

    In this way the RQ for any given tissue may be calculated. It is to be parti­cularly noted that the volume of the closed air within the bulb and the manometer should be equal in both the cases. The respirometers should be of the same size and volume and equal volumes of tissue should be taken in each case.

    Since KOH vial is kept in one bulb only, a similar vial containing equal quantity of brine solution may be kept in the other bulb so that same volume of air is present in both the bulbs. Temperature greatly affects the volume of gases and hence both respirometers should be placed at the same temperature.

    Experiment # 8

    Demonstration of Liberation of Heat Energy During Respiration:

    Three thermos-flasks are taken. One contains water-soaked seeds, second dry seeds and the third boiled seeds (dead) and all the lots are of equal weight. The mouths of the thermos-flasks are corked through which passes a thermometer in each flask so that the bulb of the thermometer remains within the seeds. The flasks are left for 24 hours.

    Rise of temperature is observed in all the cases.

    The rise in temperature of the flasks containing soaked seeds indicates that heat is produced during respiration of seeds. Tempe­rature remains nearly unchanged in dry seeds and completely unchanged in case of boiled seeds. This is because the respiration of dry seeds is almost negligible and in case of boiled seeds nil.

    N.B. The mathematical evaluation of heat loss during respiration of one gram mole of glucose is as follows. Every mole of ATP formed from ADP and phosphate requires about 12 K. Cal. of energy. But complete combustion or chemical oxidation of one mole of glucose yields 684 K. Cal. of energy as heat. Therefore, 684—456 = 228 K. Cal. is lost as heat energy. Thus in aerobic respiration of each mole of glucose about = 67 % energy of each glucose mole is carried into 38 mole of ATP. This is called as the “efficiency of energy conservation” in aerobic respiration.

    Experiment # 9

    Demonstration of Loss of Weight in Respiration:

    About 50 seeds are surface sterilised with, 1% HgCL2, washed thoroughly and 5 such seeds are separately placed in ten petridishes containing soaked filter paper. The pertidishes are kept in dark and seeds are allowed to germinate. The fresh and dry weights of each lot of seedlings are taken every alternate day.

    The percentage loss in dry weight is calculated in each case and the results are graphically plotted taking loss in weight as ordinate and days as abscissa.

    Respiration is a catabolic process which takes place by breaking down of stored carbohydrate and liberating CO2, H2O and energy. Since the seedlings are grown in dark the anabolic process, i.e., photosynthesis, cannot take place and as a result of which loss in weight occurs as the plants grow.

    Experiment # 10

    Effect of Wounding on Respiration:

    Experiment can be conveniently performed either with Warburg’s method or Pettenkoffer’s method. Two lots of potato tubers are taken one lot is slightly larger than the other.

    The larger potatoes are peeled off and surface area is made comparable with the other lot. Approximately the equal weights of these two lots are washed and dried and the two samples are placed in two respiration chambers of the Pettenko­ffer’s apparatus or in the flasks of Warburg’s apparatus. The rate of respiration is determined in both the cases at a constant temperature.

    Respiration rates in millilitres of CO2 per gram dry tissue per hour are plotted. The dry weights of the samples are determined at the end of the experiment.

    Injury to a given plant tissue often causes the respiratory activity to increase. Generally, in case of injured plant tissues the rate of respiration increases for the time being and this increase gradually rises to a maximum point and then the rate decreases.

    Wounding generally initiates meristematic activity in the area of the wound, resulting in the develop­ment of “wound callus”. It has been shown that a considerable increase in sugar content (about 70%) around the injured cells takes place.

    Perhaps the increase in respiration due to wounding is caused by increased avail­ability of respiratory substrate and meristematic cells.

    Experiment # 11

    Effect of Pre-treatment with Carbon Dioxide on the Rate of Respiration:

    Equal weights of three lots of potato tubers are taken. Two lots are taken in two petridishes placed on, two ground glass plates and each is covered with a 1litre bell jar having an outlet at the top (Figure 25).

    The lower rims of the bell jars arc made air-tight with grease. The bell jars are partially evacuated with the help of a suction pump. Now CO2 is passed from a Woulfe’s bottle (CaCO2+HCL) into one bell jar for 10 minutes and to the other for 20 minutes. The outlets are then closed and tubers are kept in these atmospheres for about three hours.

    The control lot is also placed under a bell jar for the same period of time under normal atmosphere. A centrally placed thermometer records the temperature. After the stipulated time the tubers are taken out and rates of res­piration are determined with the help of Ganong’s respirometer.

    The rate of respiration in each case is graphically plotted and compared.

    Increasing concentration of CO2 has a definite repressing effect on respiration. Since the direct measurement of respiration rate in terms of CO2 evolution and simultaneous increase in CO2 concentration is difficult with ordinary apparatus here the effect of pre-treatment of CO2 is studied. CO2 input raises the internal concentration of CO2 considerably and limits respiration by its toxic effect.

    Experiment # 12

    Effect of Moisture Content on Respiration of Grains:

    About 300 grams of dehusked rice or wheat seeds are taken in a beaker and dried in an oven at 40°C for 24 hours. These seeds are divided into 5 lots. The initial moisture content of one lot is deter­mined. Water is added to the other 4 lots and each lot is soaked for 5, 10, 15 and 30 minutes separately.

    The moisture content of each lot is then determined by taking a portion of seed from each lot. Equal weights of seeds from each lot including un-soaked one are taken in Ganong’s respirometer and rate of respiration is measured.

    The rate of respiration is expressed in terms of millilitre of CO2 evolved per gram of dry seeds per hour and graphically plotted in each case.

    Within certain limits, moisture content of tissue affects its respiratory rate. In dormant seeds water content is generally less than 10% and their respiratory rate is very slow.

    Seed, when come in contact with moisture imbibe water and swell and their respiratory rate gradually increases. The moisture content of the tissue increases the amount of soluble respiratory substrate and also the activity of the protoplasm by the enzymes.

    Experiment # 13

    Effect of Food Supply on Respiration:

    One lot of leaf sample is collected from rice plants which were previously kept in dark for 24 hours and another lot is collected from rice plants previously kept in light for 24 hours. The rate of respiration of equal weights of each sample is measured in a Pettenkoffer’s tube or in Ganong’s respirometer. The dry weight of each sample is determined.

    The results are expressed as millilitre of CO2 evolved per gram of dry tissue per hour.

    The rate of respiration of a given tissue is governed by the concentration of the soluble respirable substrates. Since organic materials are oxidised during respiration, the amount and kind of materials present in the cells appreciably affect both the rate and course of respiration.

    The rate of respiration increases due to increased carbohydrate production as a result of photosynthesis and decreases due to lower carbohydrate content in the dark. Thus the concentration of respirable substrate may limit the rates of CO2 production or O2 uptake.

    N.B. Increased respiration is also observed when various sugars (Especially sucrose, glucose, fructose or maltose) are supplied to floating leaves or other tissues in solutions.

    Experiment # 14

    Determination of Rates of Respiration and Nature of Substrates by McDougal Respiroscopes:

    This is a simple apparatus fixed in a wooden frame (Figure 26) which consists of a pair of vertical tubes. The upper end of the tube is wider than the lower end which is graduated. The lower end of the graduated tube dips into a beaker containing brine solution.

    The upper bulb-like wider ends of the respiroscopes are closed by means of corks through which pass two small tubes having bent ends. The upper ends, i.e., the outside ends of the bent tubes are fitted with stopcocks.

    From the lower bent ends of the two tubes, two small vials one containing KOH pellets hang within the bulbs. Equal amounts of germinated starchy or fatty seeds (seed coat removed) are placed within the wider bulbs of the respiroscope on a soaked cotton plug. A thermometer may be centrally placed to record temperature.

    The stopcock is then turned to make connection with atmospheric air. The brine solution rises through the graduated end of the respiroscope and becomes stationary at the brine level of the beakers. The stopcock is closed and the level of brine solution in both the tubes is recorded. The rise of brine columns in the tubes is recorded at an interval of 15 minutes for 2 hours.

    The rate of rise of water column indicates the rate of respiration.

    The partial vacuum created due to absorption of O2dur­ing respiration cannot be filled up by CO2 released and this vacuum is then filled up by the brine solution. The volume of CO2evolved during respiration by starchy seeds is equal to the volume of O2consumed (RQ = 1). But in case of fatty seeds the volume of CO2 liberated is less than the volume of O2 used up (RQ, < 1).

    Hence the rate of respiration as measured in terms of O2consumption is less in case of fatty seeds than starchy seeds. The depression (when RQ,> 1) or elevation (when RQ, < 1) column of brine solution in the control McDougal res­piroscope (without KOH) indicates the nature of Substrate. If the brine level is stationary (RQ, = 1) then the substrate is carbohydrate.

    Experiment # 15

    Respiration of Roots of Intact Plants:

    The adventitious roots of rice or wheat plants are care­fully washed with water and placed in a bottle containing water which is made slightly alkaline with dilute NaOH solution. This is coloured pink with addition of a few drops of phenolphthalein.

    A second bottle b prepared in the same way, stoppered tightly and left without a plant. Both the bottles are allowed to stand in diffused light and the solutions are examined from time to time and change in colour is carefully noted in each case.

    The pink colour of the solution in the bottle containing the plant gradually fades and ultimately becomes colourless after a consider­able time. The solution of the other bottle remains as such.

    During respiration of roots CO2 is released which is converted to H2CO3when comes in contact with water. This acid neu­tralises the dilute NaOH solution and pink colour of the solution fades.

    When a portion of this neutralised solution is gently boiled for few minutes the pink colour reappears because CO2comes off on boiling leaving the solution alkaline again.

    Experiment # 16

    Demonstration of Continuity of Intercellular Spaces:

    A conical flask is taken and half-filled with water. The mouth of the flask is fitted with a cork having two holes, through one of which is inserted a long petioled leaf of arum (Colocasia) so that the cut end of the petiole remains well under water.

    Through another hole is inserted a bent glass tube which is connected to a suction pump. The end of this bent tube remains well above the water surface. All connections are made air-tight. The air within the flask is drawn out by the suction pump.

    Bubbles are seen to come out from the cut end of the petiole into the water of the flask.

    The experiment shows that as the air is sucked, the at­mospheric air enters through the stomata of the leaf and through the inter­cellular spaces ultimately comes out through the cut end of the petioles. This shows that stomata and intercellular spaces form continuity and are involved in gaseous exchange.


    Can fermentation and aerobic respiration occur at the same time? - Biology

    Many cells are unable to carry out respiration because of one or more of the following circumstances:

    1. The cell lacks a sufficient amount of any appropriate, inorganic, final electron acceptor to carry out cellular respiration.
    2. The cell lacks genes to make appropriate complexes and electron carriers in the electron transport system.
    3. The cell lacks genes to make one or more enzymes in the Krebs cycle.

    Whereas lack of an appropriate inorganic final electron acceptor is environmentally dependent, the other two conditions are genetically determined. Thus, many prokaryotes, including members of the clinically important genus Streptococcus, are permanently incapable of respiration, even in the presence of oxygen. Conversely, many prokaryotes are facultative, meaning that, should the environmental conditions change to provide an appropriate inorganic final electron acceptor for respiration, organisms containing all the genes required to do so will switch to cellular respiration for glucose metabolism because respiration allows for much greater ATP production per glucose molecule.

    If respiration does not occur, NADH must be reoxidized to NAD + for reuse as an electron carrier for glycolysis, the cell’s only mechanism for producing any ATP, to continue. Some living systems use an organic molecule (commonly pyruvate) as a final electron acceptor through a process called fermentation. Fermentation does not involve an electron transport system and does not directly produce any additional ATP beyond that produced during glycolysis by substrate-level phosphorylation. Organisms carrying out fermentation, called fermenters, produce a maximum of two ATP molecules per glucose during glycolysis. Table 1 compares the final electron acceptors and methods of ATP synthesis in aerobic respiration, anaerobic respiration, and fermentation. Note that the number of ATP molecules shown for glycolysis assumes the Embden-Meyerhof-Parnas pathway. The number of ATP molecules made by substrate-level phosphorylation (SLP) versus oxidative phosphorylation (OP) are indicated.

    Microbial fermentation processes have been manipulated by humans and are used extensively in the production of various foods and other commercial products, including pharmaceuticals. Microbial fermentation can also be useful for identifying microbes for diagnostic purposes.

    Fermentation by some bacteria, like those in yogurt and other soured food products, and by animals in muscles during oxygen depletion, is lactic acid fermentation. The chemical reaction of lactic acid fermentation is as follows:

    Bacteria of several gram-positive genera, including Lactobacillus, Leuconostoc, and Streptococcus, are collectively known as the lactic acid bacteria (LAB), and various strains are important in food production. During yogurt and cheese production, the highly acidic environment generated by lactic acid fermentation denatures proteins contained in milk, causing it to solidify. When lactic acid is the only fermentation product, the process is said to be homolactic fermentation such is the case for Lactobacillus delbrueckii and S. thermophiles used in yogurt production. However, many bacteria perform heterolactic fermentation, producing a mixture of lactic acid, ethanol and/or acetic acid, and CO2 as a result, because of their use of the branched pentose phosphate pathway instead of the EMP pathway for glycolysis. One important heterolactic fermenter is Leuconostoc mesenteroides, which is used for souring vegetables like cucumbers and cabbage, producing pickles and sauerkraut, respectively.

    Lactic acid bacteria are also important medically. The production of low pH environments within the body inhibits the establishment and growth of pathogens in these areas. For example, the vaginal microbiota is composed largely of lactic acid bacteria, but when these bacteria are reduced, yeast can proliferate, causing a yeast infection. Additionally, lactic acid bacteria are important in maintaining the health of the gastrointestinal tract and, as such, are the primary component of probiotics.

    Another familiar fermentation process is alcohol fermentation, which produces ethanol. The ethanol fermentation reaction is shown in Figure 1. In the first reaction, the enzyme pyruvate decarboxylase removes a carboxyl group from pyruvate, releasing CO2 gas while producing the two-carbon molecule acetaldehyde. The second reaction, catalyzed by the enzyme alcohol dehydrogenase, transfers an electron from NADH to acetaldehyde, producing ethanol and NAD + . The ethanol fermentation of pyruvate by the yeast Saccharomyces cerevisiae is used in the production of alcoholic beverages and also makes bread products rise due to CO2 production. Outside of the food industry, ethanol fermentation of plant products is important in biofuel production.

    Figure 1. The chemical reactions of alcohol fermentation are shown here. Ethanol fermentation is important in the production of alcoholic beverages and bread.

    Beyond lactic acid fermentation and alcohol fermentation, many other fermentation methods occur in prokaryotes, all for the purpose of ensuring an adequate supply of NAD + for glycolysis (Table 2). Without these pathways, glycolysis would not occur and no ATP would be harvested from the breakdown of glucose. It should be noted that most forms of fermentation besides homolactic fermentation produce gas, commonly CO2 and/or hydrogen gas. Many of these different types of fermentation pathways are also used in food production and each results in the production of different organic acids, contributing to the unique flavor of a particular fermented food product. The propionic acid produced during propionic acid fermentation contributes to the distinctive flavor of Swiss cheese, for example.

    Several fermentation products are important commercially outside of the food industry. For example, chemical solvents such as acetone and butanol are produced during acetone-butanol-ethanol fermentation. Complex organic pharmaceutical compounds used in antibiotics (e.g., penicillin), vaccines, and vitamins are produced through mixed acid fermentation. Fermentation products are used in the laboratory to differentiate various bacteria for diagnostic purposes. For example, enteric bacteria are known for their ability to perform mixed acid fermentation, reducing the pH, which can be detected using a pH indicator. Similarly, the bacterial production of acetoin during butanediol fermentation can also be detected. Gas production from fermentation can also be seen in an inverted Durham tube that traps produced gas in a broth culture.

    Microbes can also be differentiated according to the substrates they can ferment. For example, E. coli can ferment lactose, forming gas, whereas some of its close gram-negative relatives cannot. The ability to ferment the sugar alcohol sorbitol is used to identify the pathogenic enterohemorrhagic O157:H7 strain of E. coli because, unlike other E. coli strains, it is unable to ferment sorbitol. Last, mannitol fermentation differentiates the mannitol-fermenting Staphylococcus aureus from other non–mannitol-fermenting staphylococci.

    Table 2. Common Fermentation Pathways
    Pathway End Products Example Microbes Commercial Products
    Acetone-butanol-ethanol Acetone, butanol, ethanol, CO2 Clostridium acetobutylicum Commercial solvents, gasoline alternative
    Alcohol Ethanol, CO2 Candida, Saccharomyces Beer, bread
    Butanediol Formic and lactic acid ethanol acetoin 2,3 butanediol CO2 hydrogen gas Klebsiella, Enterobacter Chardonnay wine
    Butyric acid Butyric acid, CO2, hydrogen gas Clostridium butyricum Butter
    Lactic acid Lactic acid Streptococcus, Lactobacillus Sauerkraut, yogurt, cheese
    Mixed acid Acetic, formic, lactic, and succinic acids ethanol, CO2, hydrogen gas Escherichia, Shigella Vinegar, cosmetics, pharmaceuticals
    Propionic acid Acetic acid, propionic acid, CO2 Propionibacterium, Bifidobacterium Swiss cheese

    Think about It

    • When would a metabolically versatile microbe perform fermentation rather than cellular respiration?

    Identifying Bacteria by Using API Test Panels

    Identification of a microbial isolate is essential for the proper diagnosis and appropriate treatment of patients. Scientists have developed techniques that identify bacteria according to their biochemical characteristics. Typically, they either examine the use of specific carbon sources as substrates for fermentation or other metabolic reactions, or they identify fermentation products or specific enzymes present in reactions. In the past, microbiologists have used individual test tubes and plates to conduct biochemical testing. However, scientists, especially those in clinical laboratories, now more frequently use plastic, disposable, multitest panels that contain a number of miniature reaction tubes, each typically including a specific substrate and pH indicator. After inoculation of the test panel with a small sample of the microbe in question and incubation, scientists can compare the results to a database that includes the expected results for specific biochemical reactions for known microbes, thus enabling rapid identification of a sample microbe. These test panels have allowed scientists to reduce costs while improving efficiency and reproducibility by performing a larger number of tests simultaneously.

    Many commercial, miniaturized biochemical test panels cover a number of clinically important groups of bacteria and yeasts. One of the earliest and most popular test panels is the Analytical Profile Index (API) panel invented in the 1970s. Once some basic laboratory characterization of a given strain has been performed, such as determining the strain’s Gram morphology, an appropriate test strip that contains 10 to 20 different biochemical tests for differentiating strains within that microbial group can be used. Currently, the various API strips can be used to quickly and easily identify more than 600 species of bacteria, both aerobic and anaerobic, and approximately 100 different types of yeasts. Based on the colors of the reactions when metabolic end products are present, due to the presence of pH indicators, a metabolic profile is created from the results (Figure 2). Microbiologists can then compare the sample’s profile to the database to identify the specific microbe.

    Figure 2. The API 20NE test strip is used to identify specific strains of gram-negative bacteria outside the Enterobacteriaceae. Here is an API 20NE test strip result for Photobacterium damselae ssp. piscicida.

    Clinical Focus: Alex, Part 2

    This example continues Alex’s story that started in Energy Matter and Enzymes.

    Many of Alex’s symptoms are consistent with several different infections, including influenza and pneumonia. However, his sluggish reflexes along with his light sensitivity and stiff neck suggest some possible involvement of the central nervous system, perhaps indicating meningitis. Meningitis is an infection of the cerebrospinal fluid (CSF) around the brain and spinal cord that causes inflammation of the meninges, the protective layers covering the brain. Meningitis can be caused by viruses, bacteria, or fungi. Although all forms of meningitis are serious, bacterial meningitis is particularly serious. Bacterial meningitis may be caused by several different bacteria, but the bacterium Neisseria meningitidis, a gram-negative, bean-shaped diplococcus, is a common cause and leads to death within 1 to 2 days in 5% to 10% of patients.

    Given the potential seriousness of Alex’s conditions, his physician advised his parents to take him to the hospital in the Gambian capital of Banjul and there have him tested and treated for possible meningitis. After a 3-hour drive to the hospital, Alex was immediately admitted. Physicians took a blood sample and performed a lumbar puncture to test his CSF. They also immediately started him on a course of the antibiotic ceftriaxone, the drug of choice for treatment of meningitis caused by N. meningitidis, without waiting for laboratory test results.

    • How might biochemical testing be used to confirm the identity of N. meningitidis?
    • Why did Alex’s doctors decide to administer antibiotics without waiting for the test results?

    We’ll return to Alex’s example in later pages.

    Key Concepts and Summary

    • Fermentation uses an organic molecule as a final electron acceptor to regenerate NAD + from NADH so that glycolysis can continue.
    • Fermentation does not involve an electron transport system, and no ATP is made by the fermentation process directly. Fermenters make very little ATP—only two ATP molecules per glucose molecule during glycolysis.
    • Microbial fermentation processes have been used for the production of foods and pharmaceuticals, and for the identification of microbes.
    • During lactic acid fermentation, pyruvate accepts electrons from NADH and is reduced to lactic acid. Microbes performing homolactic fermentation produce only lactic acid as the fermentation product microbes performing heterolactic fermentation produce a mixture of lactic acid, ethanol and/or acetic acid, and CO2.
    • Lactic acid production by the normal microbiota prevents growth of pathogens in certain body regions and is important for the health of the gastrointestinal tract.
    • During ethanol fermentation, pyruvate is first decarboxylated (releasing CO2) to acetaldehyde, which then accepts electrons from NADH, reducing acetaldehyde to ethanol. Ethanol fermentation is used for the production of alcoholic beverages, for making bread products rise, and for biofuel production.
    • Fermentation products of pathways (e.g., propionic acid fermentation) provide distinctive flavors to food products. Fermentation is used to produce chemical solvents (acetone-butanol-ethanol fermentation) and pharmaceuticals (mixed acid fermentation).
    • Specific types of microbes may be distinguished by their fermentation pathways and products. Microbes may also be differentiated according to the substrates they are able to ferment.

    Multiple Choice

    Which of the following is the purpose of fermentation?

    1. to make ATP
    2. to make carbon molecule intermediates for anabolism
    3. to make NADH
    4. to make NAD +

    Which molecule typically serves as the final electron acceptor during fermentation?

    Which fermentation product is important for making bread rise?

    Which of the following is not a commercially important fermentation product?

    Fill in the Blank

    The microbe responsible for ethanol fermentation for the purpose of producing alcoholic beverages is ________.

    ________ results in the production of a mixture of fermentation products, including lactic acid, ethanol and/or acetic acid, and CO2.

    Fermenting organisms make ATP through the process of ________.

    Matching

    Match the fermentation pathway with the correct commercial product it is used to produce:



Comments:

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  5. Etienne

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  6. Bane

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