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Do acetic acid bacteria use the electron transport chain when converting ethanol to acetic acid?

Do acetic acid bacteria use the electron transport chain when converting ethanol to acetic acid?


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Do acetic bacteria use the electron transport chain when converting ethanol to acetic acid?

And is wikipedia inconsistent here in its definition of fermentation. It says fermentation

Fermentation takes place when the electron transport chain is unusable

(and this is consistent with (academic) microbiology books searchable on google books). Wikipedia defines acetic acid bacteria as

produce acetic acid during fermentation.

also

Some genera, such as Acetobacter, can oxidize ethanol to carbon dioxide and water using Krebs cycle enzymes. Other genera, such as Gluconobacter, do not oxidize ethanol, as they do not have a full set of Krebs cycle enzymes.

I read in microbiology books on google books, that acetic acid bacteria use oxygen as the terminal electron acceptor, and have a respiratory mechanism.

Brewing Microbiology edited by Fergus Priest p165

Acetobactor spp. possess a respiratory mechanism… Acetobactor spp. are obligately aerobic with a respiratory metabolism (O2 as the terminal electron acceptor)

So is wikipedia wrong to describe acetic acid bacteria as using fermentation?

I see mention of krebs cycle though, which I understand to be associated with respiration rather than fermentation. Also in this reaction/process :

$C_2H_6Ohspace{1mm}(ethanol) + O_2 ightarrow C_2H_4O_2hspace{1mm}(acetichspace{1mm}acid) + H_2O$

I see water mentioned but not carbon dioxide(if it had both I'd say that really looks like respiration, though it's being called oxidative fermentation). So I still can't really see whether it's respiration or fermentation, though it's being called fermentation (oxidative fermentation), yet there is the mention of krebs cycle enzymes (krebs cycle being associated with respiration), so it makes me unsure whether the electron transport chain is used or not.


Yes

I find it somewhat ironic that in a response a recent post from the poster concerning itself with the precise definition of 'fermentation' I argued that this was a semantic question because of the use of the term in the English language before any biochemistry was known. I now discover that the term is used in an even looser sense than I was aware of - in an aerobic context. The conversion of ethanol to acetic acid by acetic acid bacteria is apparently referred to as oxidative fermentation as explained in this extract from a paper in The Journal of Bacteriology:

Acetic acid bacteria are obligate aerobes that belong to the α-Proteobacteria and have a strong ability to oxidize ethanol, sugar alcohols, and sugars into their corresponding organic acids. Such oxidation reactions are traditionally called oxidative fermentation, since they involve incomplete oxidation of these compounds. These bacteria accumulate the corresponding incomplete oxidation products in large quantities in their surrounding environment.

The biochemistry is summarized by Gómez-Manzo et al.:

Ethanol fermentation by acetic acid bacteria is carried out by two sequential reactions catalyzed by pyrroloquinolinequinone (PQQ)-dependent alcohol dehydrogenase enzymes (ADH) and aldehyde dehydrogenase (ALDH), which are located in the cytoplasmic membrane [6] and transfer electrons to ubiquinone Q10 [7]. PQQ-ADH is a periplasmic quinohemoprotein-cytochrome c complex and catalyzes the first step of ethanol oxidation by transferring electrons to Q10 and producing acetaldehyde which usually is the substrate for another enzyme (ALDH), and converted to acetic acid during the second step of ethanol fermentation.

The fact that the reduced pyrroloquinolinequinone is re-oxidised by ubiquinone, indicates that the conversion of ethanol to acetic acid requires the electron transport chain in the bacterial membrane to continue, and that the ultimate electron acceptor is oxygen.


I do not know about the prokaryotic mechanism for ethanol metabolism. However, in eukaryotes, the metabolism of ethanol does indeed eventually show an effect in the ETC.

When ethanol is metabolised in eukaryotes, alcohol dehydrogenase will remove a proton with the help of NAD+ to create acetate and NADH. Of course, NADH is used within the ETC. So basically, the metabolism of ethanol serves as one of many ways a cell can balance its redox potential.

Some organisms fermentative pathways are inhibited by the presence of oxygen, and some are not. I can not tell you with certainty whether or not this is the case for you in particular. But, I would define a fermentative process as, a process which does not result in the complete oxidation of a carbon-substrate.


Do acetic acid bacteria use the electron transport chain when converting ethanol to acetic acid? - Biology

Metabolism -- The sum of all chemical reaction within a cell. It can also be described as catabolism + anabolism .

Chemical Reactions
Some reactions require energy. Energy must be added in order to make these reactions happen and the product(s) will be at a higher energy level than the reactants. In metabolism, many anabolic reactions fall into this category. Anabolic reactions require energy. Catabolic reactions release energy.

Not all energetically favored reactions are spontaneous. Many times some energy of activation needs to be added. For example, paper (cellulose = C 6 H 12 O 6 ) exists stably in the presence of oxygen. Even though the rapid oxidation of the cellulose to form CO 2 , H2O and C is energetically favored, the paper won't burn (burning = the rapid oxidation of cellulose) unless activation energy (heat) is applied.

I. ENZYMES
In the cell, the energy needed to drive anabolic reactions as well as the activation energy needed to get many catabolic reactions going cannot be directly applied as heat. Instead, cells use enzymes to lower the amount of energy needed to cause the reactions to occur. Thus enzymes are called catalysts because the facilitate reactions and speed them up but they don't enter into the reactions.

Enzymes lower the activation energy of reactions because enzymes are able to (1) bind to the reactants (substrate), (2) force the reactants (substrate molecules) very close to each other and (3) bend the substrate molecules and destabilize their electron configurations. This makes the molecules unstable and reactive.

  • The place on the enzyme where substrate binds is called the substrate binding site or the active site of the enzyme. Allosteric site is a site other than the active site.
  • Apoenzyme = the protein portion
  • Cofactors = are non-protein atoms or molecules which bind to the apoenzyme. They are divided into organic molecules = coenzymes, and inorganic elements = metal ions.
  • Coenzymes= NAD+ (nicotinamide adenine dinucleotide), FAD (flavin adenine dinucleotide), CoA (coenzyme A)
  • Metal ions = Iron, copper, calcium, zinc, magnesium.
  • Holoenzyme = Apoenzyme + Cofactor

III. Factor affecting enzyme function: (don't forget saturation!)
1) pH
2) Temperature
3) Substrate concentration
4) Enzyme concentration

IV. Enzyme Inhibition:
a) Competitive Inhibition: A molecule with similar structure to the normal substrate can occupy (and block) the enzyme's active site. Can be reversed by adding more substrate. E.g. folic acid synthetase binds PABA ---> folic acid. The drug sulfanilamide has a chemical structure very similar to PABA and the drug will bind to the active site of the enzyme. Folic acid synthetase however is incapable of converting sulfanilamide into anything.

b) Non-competive Inhibition: Inhibitors (e.g. lead or other metals) can bind to the allosteric site changing the shape of the enzyme. Now, the active site is different and can't bind to the substrate.

ENERGY FLOW IN METABOLISM
Energy in metabolism often flows in terms of electrons. If electrons ARE LOST, this is called oxidation. If electrons ARE GAINED, this is called reduction. Oxidation is coupled to reduction that is, if something gets oxidized, then something else gets reduced (remember the first and second laws of thermodinamics!).

In most of the oxidations and reductions which we will study, electrons (e-) will be moved with protons (H+). Watching the hydrogens therefore provides a convenient way to tell if a molecule has been oxidized or reduced.

Also, in many of the oxidation-reduction reactions we will look at, the molecule nicotinamide adenine dinucleotide (NAD) which serves as an electron-shuttle. NAD can become REDUCED to NADH 2 , and then carry the electrons to some other reaction and become OXIDIZED back to NAD. In other words, NAD can pick up electrons from one reaction and carry them to another.

Note that when a molecule gets OXIDIZED IT LOSES ENERGY. Also, the more reduced a molecule is the more energy it contains. (See pgs 121-122, figs. 5.8, and 5.9 for descriptions of NAD and oxidation-reduction reactions.)

The ultimate goal in many instances of catabolism will be to take energy from a (food source) molecule, trap the energy and store it as ATP.

There are three ways to make ATP:

1.) Substrate level phosphorylation- where a high energy phosphate from an intermediate phosphorylated metabolic molecule gets transferred directly onto ADP in a catabolic pathway converting it to ATP.

2.) Oxidative phosphorylation - where a (food source) molecule is oxidized and the energy is extracted from the electrons by an electron transport chain.
The extracted energy is then used to make ATP by a process known as chemiosmosis.

3.) Photophosphorylation - This is seen only in cells carrying out photosynthesis. In here, light energy is used to generate electrons and then the energy is extracted from the electrons by an electron transport chain. As in oxidative phosphorylation, the extracted energy is used to make ATP by chemiosmosis.

  • Aerobic respiration, in which oxygen is the final electron acceptor
  • Anaerobic respiration, in which an inorganic molecule other than oxygen is the final electron acceptor
  • Fermentation, in which an organic molecule is the final electron acceptor, and
  • Photosynthesis, during which radiant energy is converted into chemical energy

The respiration of glucose as a fuel source occurs in 3 stages: glycolysis, Krebs cycle and electron transport chain.

Glucose + 6O 2 ----> 6CO 2 + 6H 2 O + energy

  • The partial breakdown (oxidation) of a glucose molecule (a 6-C molecule) into 2 pyruvic acid molecules (3-C molecules).
  • Uses 2 ATP's and makes 4 ATP's. So, there is a net gain of 2 ATP's
  • Makes 2 NADH 2
  • Further oxidation of carbon molecules
  • Pyruvic acid ---> acetyl-CoA + CO 2
  • Regeneration by oxaloacetic acid (4C) + acetyl-CoA (2C)
  • A lot of NADH (3-4 molecules), 2 FADH 2 produced and 6 molecules of CO 2 released.

It is a series of enzymes imbedded in a membrane. These enzymes use the membrane to set up a chemiosmotic gradient of hydrogen ions. This gradient of hydrogen ions is called a proton motive force and this force supplies the energy for an ATP synthetase.

The electron transport chain enzymes are a series of oxidation-reduction electron carrier molecules and proton pumps. These enzymes use the energy in the electrons from glycolysis and Krebs cycle to move protons against a concentration gradient to form the proton motive force.

In the mitochondria of eukaryotes, 3 pairs of protons are "pumped out" between the inner and outer mitochondrial membranes during a single run down the electron transport system and their re-entry generates the formation of 3 molecules of ATP. However, in prokaryotes, often less protons are transported across the membrane in a single run (2 pairs in E. coli) so less ATP's are generated (2 in E. coli). The principle is however, the same.

  • Metabolism of pyruvic acid and uses an organic molecule as final electron acceptor
  • Does not require oxygen
  • Regeneration of NAD+ and NADP+
  • Very little energy is produced (1 or 2 ATP's mostly from glycolysis)
  • End products are: lactic acid, CO 2 , ethanol, butanediol, propionic acid, succinic acid, acetic acid, etc.
  • No Krebs cycle or electron transport chain
  • Found only in anaerobic and facultative bacteria

Comparison between Fermentation and Aerobic respiration.

Pathways Involved Final Electron acceptor Net Products
Fermentation glycolysis organic molecules 2 ATP, CO2, ethanol, lactic acid, etc
Respiration glycolysis, Krebs cycle, electron transport chain oxygen 38 ATP, CO2, H2O

  • Remember this is for one glucose molecule!
  • NADH is going to produce 3 ATP molecules
  • FADH is going to produce 2 ATP molecules
  • Remember we only look at carbohydrate metabolism but the metabolism of fatty acids and proteins pretty much follows the same catabolic pathways.
  • We also did not look any anabolic pathways, pathways that are used to make complex molecules from simple components.

CLASSIFICATION OF ORGANISMS BY NUTRITIONAL PATTERN:

Energy is the ability to do work. Bacteria require energy for motility, active transport of nutrients into the cell, and biosynthesis of cell components such as nucleotides, RNA, DNA, proteins, peptidoglycan, etc. In other words, energy is required to drive various chemical reactions.

To get energy, bacteria (chemoheterotrophs) take energy-rich compounds such as glucose into the cell and enzymatically break them down to release their energy. Therefore, the bacterium needs a way to trap that released energy so it is not wasted as heat and store the energy in a form that can be utilized by cells. Principally, energy is trapped and stored in the form of adenosine triphosphate or ATP. Much ATP is needed for normal growth. For example, a typical growing E. coli cell must synthesize approximately 2.5 million molecules of ATP per second to support its energy needs.

  • light -- phototroph
  • oxidation -reduction of organic & inorganic compounds -- chemotroph
  • carbon dioxide -- autotroph (Self -feeders)
  • organic compounds -- heterotroph

Chemoheterotrophs= energy and carbon from organic molecules
Chemoautotrophs= energy from reduced inorganic compounds and CO 2 as source of carbon.


6.2: Fermentation

  • Contributed by OpenStax
  • General Biology at OpenStax CNX
  • Define fermentation and explain why it does not require oxygen
  • Describe the fermentation pathways and their end products and give examples of microorganisms that use these pathways
  • Compare and contrast fermentation and respiration

There are two mechanisms by which chemoheterotrophs can generate ATP: respiration and fermentation. Although respiration relies on the generation of a proton gradient and ATP synthesis by oxidative phosphorylation, ATP synthesis in fermentation is entirely through substrate-level phosphorylation in metabolic pathways. In general, the amount of ATP produced through fermentation is less than respiration, but there are situations where fermentation is necessary or preferable. Many prokaryotes, such as E. coli, 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 respiration because respiration allows for much greater ATP production. Whereas lack of an appropriate inorganic final electron acceptor is environmentally dependent, some organisms lack the ability to respire altogether. Many prokaryotes, including members of the clinically important genera Streptococcus and Clostridium, rely entirely on fermentation for ATP generation.

If respiration does not occur, NADH must be reoxidized to NAD + for reuse as an electron carrier for glycolysis and other catabolic pathways to continue. Some living systems use a metabolite produced through the cell's metabolite (such as pyruvate) as a final electron acceptor through a process called fermentation. Because all the NADH produced must be reoxidized to NAD+, the net NADH of any fermentation pathway must be zero (0). Considering that the purpose of fermentation is to generate ATP, there must also be a net gain of ATP in these metabolic pathways.

Fermentation does not involve an electron transport chain and does not directly produce any additional ATP beyond that produced during glycolysis by substrate-level phosphorylation. Organisms carrying out fermentation typically produce a maximum of two ATP molecules per glucose during glycolysis. Table (PageIndex<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.

Electron transport and chemiosmosis (OP):

Electron transport and chemiosmosis (OP):

In all bacterial fermentations, at least one of the waste products produced is an organic acid. This feature of bacterial fermentations is frequently exploited in metabolic tests used to identify bacteria. 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&ndashmannitol-fermenting staphylococci.

The simplest fermentation, which is used by some bacteria, like those in yogurt and other soured food products, and by animals in muscles during oxygen depletion, is homolactic or lactic acid fermentation (Figure (PageIndex<1>). In homolactic fermentation the electrons on NADH produced during glycolysis are reoxidized to NAD+ by donating their electrons to the end product of glycolysis, pyruvate. The resulting waste product is lactate (lactic acid).

Figure (PageIndex<1>): Homolactic (lactic acid) fermentation. Note that the 2 NADH produced in glycolysis are reoxidized to NAD+ when their electrons are added to pyruvate to make the waste product lactate (lactic acid) (2021 Jeanne Kagle)

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 by yeast, which produces ethanol. The ethanol fermentation reaction is shown in Figure (PageIndex<2>). You may notice that unlike bacterial fermentations this fungal (eukaryotic) fermentation does not produce an acid as a waste product. 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 (PageIndex<2>): 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 microbes, all for the purpose of ensuring an adequate supply of NAD + for glycolysis (Table (PageIndex<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.

In addition to fermentation ability, 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.

Table (PageIndex<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

When would a metabolically versatile microbe perform fermentation rather than 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&rsquos 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 (PageIndex<2>)). Microbiologists can then compare the sample&rsquos profile to the database to identify the specific microbe.

Figure (PageIndex<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.


Biology Question Bank – 38 MCQs on “Cell Respiration” – Answered!

38 Questions with Answers and Explanations on “Cell Respiration” for Biology Students.

1. Incomplete oxidation of glucose into pyruvic acid with several intermediate steps is known as

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Answer and Explanation:

1. (b): Glycolysis is the biochemical change in which one molecule of glucose is converted into 2 molecules of pyruvic acid with the involvement of ten enzymes. It is independent of oxygen and is common to both aerobic and anaerobic condition. It takes place in cytoplasm and all the reactions are reversible.

All the intermediates of glycolysis are not converted into pyruvic acid. Some of them build back the carbohydrates and the phenomenon is called as oxidative anabolism. TCA cycle and Krebs cycle are synonym where the pyruvic acid of glycolysis is utilized to form CO2. HMS is hexose monophosphate shunt or pentose phospate pathway which is an alternative pathway of glycolysis.

2. NADP + is reduced to NADPH is

Answer and Explanation:

2. (a): HMP pathway generates NADPH molecule which are used as reductants in biosynthetic process under conditions when NADPH molecules are not generated by photosynthesis. It is, therefore, important in non- photosynthetic tissues such as in differentiating tissues, generating seeds and during periods of darkness. Production of NADPH is not linked to ATP generation in pentose phosphate pathway.

4. End product of glycolysis is

Answer and Explanation:

4. (b): In glycolytic cycle, each molecule of glucose (a hexose sugar) is broken down in step wise biochemical reactions under enzymatic control into two molecules of pyruvic acids. It takes place is cytosol.

(a) CO2 produced to substrate consumed

(b) CO2 produced to O2 consumed

(c) oxygen consumed to water produced

(d) oxygen consumed to CO2 produced.

(b) CO2 produced to O2 consumed

6. EMP can produce a total of

Answer and Explanation:

6. (b): Glycolysis is also known as EMP pathway after the names of its discoverers. Embden, Meyerhof and Paranas. In glycolysis, 8ATP are produced. 4ATP are formed from substrate level phosphorylation, out of which 2ATP are used up and net gain of 2 AT P. 6ATP are produced from oxidative phosphorylation. Hence, Total ATP produced in glycolysis is 8ATP.

7. Connecting link between glycolysis and Krebs cycle before pyruvate entering Krebs cycle is changed to

Answer and Explanation:

7. (d): End product of glycolysis is pyruvic acid which is converted into acetyl coA before entering into the Krebs cycle, which is aerobic in nature.

8. Terminal cytochrome of respiratory chain which donates electrons to oxygen is

Answer and Explanation:

8. (d): Cytochrome a3 helps in transfer of electron to oxygen. The oxygen has great affinity to accept the electrons and in presence of protons a water molecule is formed (figure).

9. Out of 36 ATP molecules produced per glucose molecule during respiration

(a) 2 are produced outside glycolysis and 34 during respiratory chain

(b) 2 are produced outside mitochondria and 34 inside mitochondria

(c) 2 during glycolysis and 34 during Krebs cycle

(d) All are formed inside mitochondria.

Answer and Explanation:

9. (b): During respiration, 36 ATP molecules are produced per glucose molecule. 2 molecules of ATP are produced outside mitochondria i.e. during glycolysis and other 34 molecules of ATP are produced inside mitochondria from Krebs cycle.

10. Link between glycolysis, Krebs cycle and P-oxidation of fatty acid or carbohydrate and fat metabolism is

Answer and Explanation:

10. (d): Krebs cycle is intimately related with fat metabolism. Dihydroxy acetone phosphate produced in glycolysis may be ‘converted into glycerol via glycerol – 3 – phosphate and vice-versa. Glycerol is important constituents of fats. After P-oxidation, fatty acids give rise to active – 2 – C units, the acetyl-CoA which may enter the Krebs cycle. Thus, Acetyl-CoA is a link between glycolysis, Krebs cycle and P- oxidation of fatty acid or carbohydrate and fat metabolism.

11. End products of aerobic respiration are

(c) carbon dioxide, water and energy

(d) carbon dioxide and energy.

Answer and Explanation:

11. (c): The food substances in living cells are oxidised in presence of oxygen, it is called aerobic respiration. Complete oxidation of food matter (1 .mole of glucose) occurs releasing 686 Kcal of energy. The ends of products formed are CO2 and H2O.

12. At a temperature above 35°C

(a) rate of photosynthesis will decline earlier than that of respiration

(b) rate of respiration will decline earlier than that of photosynthesis

(c) there is no fixed pattern

(d) both decline simultaneously.

Answer and Explanation:

12. (a): The plants can perform photosynthesis on a range of temperature, while some cryophytes can do photosynthesis at 35°C. Usually the plants can perform photosynthesis between 10°C – 40°C. The optimum temperature ranges between 25°C – 30°C. At high temperature the enzymes are denatured and hence the photosynthetic rate declines.

13. Oxidative phosphorylation is production of

(b) NADPH in photosynthesis

Answer and Explanation:

13. (c): In electron transport system the hydrogen donated by succinate is accepted by FAD which is reduced to FADH2. This hydrogen dissociates into electrons and protons and then passes through a series of carriers involving the phenomenon of oxidation and reduction. During this flow, ATP synthesis occurs at different steps and the phenomenon is called as oxidative phosphorylation.

15. Apparatus to measure rate of respiration and R.Q. is

Answer and Explanation:

15. (c): Respirometer is an instrument used for measuring R.Q and rate of respiration. The apparatus consists of a graduated tube attached at right angles to a bulbous respiratory chamber in its upper end. Desired plant material who’s R.Q is to be determined is placed in the respiratory chamber.

16. End product of citric acid cycle/Krebs cycle is

Answer and Explanation:

16. (d): The end product of glycolysis is pyruvic acid whereas acetyl CoA is the connecting link between glycolysis and Krebs cycle. The TCA cycle was first described by Krebs, 1937 as a cyclic process in which acetyl coA is oxidised to C02 and water. Acetyl CoA combines with oxalo acetic acid to form citric acid. After a series of cyclic reactions OAA is recycled back.

17. Out of 38 ATP molecules produced per glucose, 32 ATP molecules are formed from NADH/FADH2 in

(c) oxidative decarboxylation

Answer and Explanation:

17. (a): During respiratory chain, complete degradation of one glucose molecule produced 38 ATP molecules. NAD and FAD is reduced to NADH/FADH2.

18. Life without air would be

(b) free from oxidative damage

Answer and Explanation:

18. (d): Anaerobic respiration (absence of oxygen) takes place in anaerobic bacteria and in plant seeds. Anaerobic respiration occurs in the organism which can live without oxygen. In this respiration, only glycolysis takes place due to the absence of oxygen.

19. The First phase in the breakdown of glucose, in animal cell, is

20. When yeast ferments glucose, the products obtained are

21. The ultimate respiratory substrate, yielding maximum number of ATP molecules, is

Answer and Explanation:

21. (c): Glucose is the chief respiratory substrate which fields maximum number of ATP molecules. Glucose is the most common substate in glycolysis. Any other carbohydrate is first converted into glucose. During glycolysis it changes to pyruvic acid and net gain is of 2 ATP and 2 NADH2 molecules. And later on during Krebs cycle 30 molecules of ATP are produced. So a total of 38 ATP molecules are produced from 1 mol of glucose during aerobic respiration.

22. Poisons like cyanide inhibit Na + efflux and K + influx during cellular transport. This inhibitory effect is reversed by an injection of ATP. This demonstrates that

(a) ATP is the carrier protein in the transport system

(b) energy for Na + -K + exchange pump comes from ATP

(c) ATP is hydrolysed by ATPase to release energy

(d) Na + -K + exchange pump operates in the cell.

Answer and Explanation:

22. (b): Active transport is uphill movement of materials across the membrane where the solute particles move against their chemical concentration or electrochemical gradient. Hence the transport requires energy in the form of ATP. Metabolic inhibitors like cyanide inhibit absorption of solutes by lowering the rate of respiration. Consequently less ATP are formed. However, by adding ATP, active transport is facilitated.

It occurs in plants as in climacteric fruits and under cold stress. ATP synthesis does not occur. Reducing power present in reduced coenzymes is oxidised to producc heat energy. Therefore, the heat liberation pathway of terminal oxidation is cyanide resistant.

In normal aerobic respiration, the effect of cyanide poisoning can be minimised by immediate supply of ATP.

23. When one molecule of ATP is disintegrated, what amount of energy is liberated?

Answer and Explanation:

23. (c): ATP is adenosine triphosphate. It was discovered by Lohmann in 1929. It consists of a purine, adenine, a pentose sugar (ribose) and a row of three phosphates out of which the last two are attached by high energy bonds. The last phosphate bond yields an energy equivalent of 7 kcal.

However the latest concept holds that an energy equivalent of 8.15 kcal per mole is released.

24. At the end of glycolysis, six carbon compounds ultimately changes into

Answer and Explanation:

24. (c): Glycolysis or EMP pathway is the breakdown of glucose to two molecules of pyruvic acid through a series of enzyme mediated reaction releasing energy. Pyruvic acid is a 3-carbon compound. In glycolysis net gain of 2ATP and 2 NADH2 molecules occurs. It can be represented in equation form as –

2CH3COCOOH + 2 ATP + 2 NADH2

25. Which of the following products are obtained by anaerobic respiration from yeast?

Answer and Explanation:

25. (d): In the absence of O2, fermentation or anaerobic respiration occurs. The cells of yeast contain zymase complex enzyme that are capable of fermentation. It is completed in cytoplasm. In this process pyruvic acid forms ethyl alcohol and CO2.

Brewing is the name given to the combined process of preparing beverages from infusions of grains that have undergone sprouting (malting) and the fermenting of the sugary solution by yeast, whereby a portion of the carbohydrate is changed to alcohol and carbondioxide various types of beer, whisky and wine are produced. Wine is the product made by normal fermentation of the juice of ripe grapes (Vitis vinifero) using a pure culture of yeast.

26. The end products of fermentation are

Answer and Explanation:

26. (d): Fermentation or anaerobic respiration occurs in the absence of 02. It involves breakdown of organic substance particularly carbohydrates under anaerobic conditions to form ethyl alcohol and carbon dioxide. It can be represented in equation form as

27. In Krebs’ cycle, the FAD precipitates as electron acceptor during the conversion of

(a) fumaric acid to malic acid

(b) succinic acid to fumaric acid

(c) succinyl CoA to succinic acid

(d) a-ketoglutarate to succinyl CoA.

(b) succinic acid to fumaric acid

28. Which of the following is the key intermediate compound linking glycolysis to the Krebs’ cycle?

Answer and Explanation:

28. (b): During glycolysis pyruvic acid is produced from glucose and is oxidatively decarboxylated to form acetyl CoA. This formation of acetyl CoA from pyruvic acid needs a multienzyme complex and 5 essential cofactors, i.e. lipoic acid, CoA, Mg 2+ , NAD and TPP (thiamine pyrophosphate).

It results in production of 2 molecules of CO2 and 2 molecules of NADH2. This acetyl CoA enters mitochondria and is completely oxidised during Kreb’s cycle. Thus acetyl CoA acts as the linker of glycolysis and Kreb’s cycle.

29. Net gain of ATP molecules, during aerobic respiration, is

30. Organisms which obtain energy by the oxidation of reduced inorganic compounds are called

Answer and Explanation:

30. (b): Chemoautotrophs are organisms that are capable of manufacturing their organic food utilizing chemical energy released in oxidation of some inorganic substances. The process of manufacture of food in such organisms is called chemosynthesis. It includes some acrobic bacteria. Photoautotrophs obtain energy for their synthesis of food from light.

Fungi living on dead or decaying plant or animal remains and also growing on dung of herbivores are saprophytes.

31. How many ATP molecules are produced by aerobic oxidation of one molecule of glucose?

Answer and Explanation:

32. In which one of the following do the two names refer to one and the same thing?

(a) Krebs cycle and Calvin cycle

(b) tricarboxylic acid cycle and citric acid cycle

(c) citric acid cycle and Calvin cycle

(d) tricarboxylic acid cycle and urea cycle

Answer and Explanation:

32. (b): The reactions of Krebs cycle were worked out by Sir Hans Kreb, hence the name Krebs cycle. It involves many 3-C compounds such as citric acid, cis-aconitic acid and iso-citric acid etc. so it is called TCA cycle tricarboxylic acid cycle. It involves formation of citric acid as its first product so it is called citric acid cycle. It involves production of 24 ATP molecules.

33. In alcohol fermentation

(a) triose phosphate is the electron donor while acetaldehyde is the electron accept

(b) triose phosphate is the electron donor while pyruvic acid is the electron acceptor

(c) there is no electron donor

(d) oxygen is the electron acceptor

(a) triose phosphate is the electron donor while acetaldehyde is the electron accept

34. In glycolysis, during oxidation electrons are removed by

Answer and Explanation:

34. (c): During glycolysis NAD (Nicotinamide adenine dinucleotide) removes electrons from 1, 3- diphosphoglyceric acid using diphosphoglycrealdehyde dehydrogenase. NAD changes to NADH2 and this is either utilized as such in anaerobic respiration or in the presence of oxygen.

35. During which stage in the complete oxidation of glucose are the greatest number of ATP molecules formed from ADP?

(c) conversion of pyruvic acid to acetyl CoA

(d) electron transport chain.

Answer and Explanation:

35. (d): The last step of aerobic respiration is the oxidation of reduced coenzymes, i.e., NADH2 and FADH2 by molecular oxygen through FAD, ubiquinone, cyt. f, cyt. c, Cyt c,, Cyt. a and cyt. ay By oxidation of 1 molecule of NADH,, 3ATP molecules are produced and by oxidation of 1 molecule of FADH2 2 ATP molecules are produced.

In glycolysis 2 ATP molecules are produced from ADP. Further 2NADH2 produced, give 2ࡩ=6 ATP, on oxidative phosphorylation. Similarly in Kreb’s cycle 2 ATP molecules are produced. So the greatest numbers of ATP molecules are produced in the electron transport chain.

36. How many ATP molecules could maximally be generated from one molecule of glucose, if the complete oxidation of one mole of glucose to C02 and H20 yields 686 kcal and the useful chemical energy available in the high energy phosphate bond of one mole of ATP is 12 kcal?

Answer and Explanation:

36. (d): One mole of ATP liberates 12 kcal of energy. So 686 kcal will be liberated by 686/12 = 57.1 ATP molecules.

37. All enzymes of TCA cycle are located in the mitochondrial matrix except one which is located in inner mitochondrial membranes in eukaryotes and in cytosol in prokaryotes. This enzyme is

(a) isocitrate dehydrogenase

(c) succinate dehydrogenase

Answer and Explanation:

37. (c): Mitochondrion is the organelle which bears various enzymes participating in Krebs cycle. Each mitochondrion is covered by double membrane. The inner membrane is selectively permeable and forms foldings called cristae. The inner membrane bears oxysomes, enzymes of fatty acids, succinate dehydrogenase (of Krebs cycle) and electron transport system. All other enzymes of Krebs cycle are present in the mitochondrial matrix.

38. The overall goal of glycolysis, Krebs cycle and the electron transport system is the formation of

(a) ATP in one large oxidation reaction

(d) ATP in small stepwise units.

Answer and Explanation:

38. (d): Respiration is an energy liberating enzymatically controlled multistep catabolic process of step wise breakdown of organic substances (hexose sugar) inside the living cells. Aerobic respiration includes the 3 major process, glycolysis, Krebs cycle and electrons transport chain. The substrate is completely broken down to form CO2 and water. A large amount of energy is released stepwise in the form of ATP.


Acid resistance factors in the cytoplasm

Overexpression of certain enzymes in the tricarboxylic acid cycle

A study found that AAB can oxidize acetic acid into carbon dioxide and water when the ethanol substrate in culture medium is exhausted to promote secondary growth (Matsushita et al. 2016). In this process, which is known as acetic acid assimilation, acetyl-CoA synthetase (acs) catalyzes the conversion of acetate to acetyl-CoA and citrate synthase (aarA). Acetyl-CoA then enters the TCA cycle, enabling the removal of acetic acid through the TCA cycle (Ramírez-Bahena et al. 2013) (Fig. 1b). A. aceti decreases the harmful effects of acetic acid accumulation through cytoplasm acidification, showing that the cytoplasm may possess substances that can adapt to an acidic environment.

Proteomics analysis of A. pasteurianus (4% (W/V)) and Komagataeibacter spp. (> 10%(W/V)) under acid stimulation revealed various proteins that play important roles in stress response, the tricarboxylic acid cycle, cell membrane modification, and outer membrane protein and cell morphology changes (Andrés-Barrao et al. 2012). Among these proteins, overexpression of enzymes involved in the tricarboxylic acid cycle, such as citrate synthase, isocitrate dehydrogenase, dihydrolipoamide dehydrogenase, succinate dehydrogenase, succinyl-CoA and CoA transferase (Andrés-Barrao et al. 2016), further confirmed the role of the TCA cycle in acid resistance in AAB.

To analyze the specific acetic acid resistance factors in the cytoplasm of AAB, analysis of proteomes induced by acetic acid was performed to detect genes and enzymes related to acid resistance. The results revealed that three genes (aarA, aarB, and aarC) will affect acid resistance in AAB and deletion of all three genes causes acid resistance to disappear in A. aceti 1023 (Fukaya et al. 1990). CS activity was not found in aarA gene deletion mutants of A. aceti, but introduction of aarA-containing plasmids restored CS activity. These findings demonstrated that the aarA gene is citrate synthase, which is closely associated with acid resistance in A. aceti (Mullins et al. 2008). Deletion of the aarC gene in A. aceti decreases acetic acid resistance and utilization capacities, but these two functions are restored after introduction of the aarC gene. In the TCA cycle, aarC replaces succinyl-CoA synthetase and directly converts succinyl-CoA to acetyl-CoA. The appearance of the branch can decrease the cell’s metabolic need for free CoA and regulate the effects of the TCA cycle on cytoplasmic pH (Francois et al. 2006). It is speculated that the aarB gene encodes the TCA activator SixA (Mullins et al. 2008). When there is a need to decrease intracellular acetic acid concentrations, these three aar genes synergistically act together to form a complete cycle that is different from the conventional TCA cycle (Fukaya et al. 1993). Large amounts of a 100 ku protein were found in acetic acid-containing culture medium, and sequence analysis revealed that it may be aconitase. Aconitase-overexpressing A. aceti can produce high acetic acid concentrations and decrease the growth doubling time. Increased aconitase activity and acid resistance was also found to increase the acetic acid concentration by 25%, which was a significant improvement in the fermentation productivity of acetic acid (Nakano et al. 2004).

The above studies confirmed that increasing the activity of one or more enzymes in the TCA cycle such as citrate synthase and aconitase will lead to rapid consumption of acetic acid or elimination of toxicity due to entry of acetic acid into the cytoplasm, causing intracellular acetic acid to be maintained at a low level and increasing acetic acid resistance.

Heat stress proteins

Universal stress mechanisms are regulated by stress proteins known as molecular chaperones or chaperone proteins. HSPs are typical stress proteins that ensure correct folding of synthesized proteins in adverse environments and prevent intracellular protein denaturation (Hartl and Hayer-Hartl 2002).

GroES/L and DnaK/J are two common universal stress protein systems in AAB that are able to respond to many types of adverse environments (Yukphan et al. 2009). The HSP GroEL is significantly upregulated in A. aceti during batch feeding and continuous fermentation (Steiner and Sauer 2001). The transcript level of the groESL gene in A. aceti IFO 3283 was upregulated by heat, ethanol, and acetic acid. Furthermore, intracellular overexpression of the groESL gene can increase resistance to the aforementioned factors, showing that the groESL gene is related to resistance to adverse environments in AAB (Okamoto-Kainuma et al. 2002). Overexpression corresponding genes of intracellular grpE and dnaKJ increased resistance towards the fermentation environment in AAB (Ishikawa et al. 2010 Okamoto-Kainuma et al. 2004). Employing two-dimensional electrophoresis to conduct a comprehensive study of intracellular protein levels in A. pasteurianus LMG 1262 T during acetic acid fermentation, it was found that fermentation increased the protein expression levels of GrpE, DnaK, DnaJ, GroES, GroEL, and ClpB to varying extents, with the expression level of GrpE being increased by 9.42 times compared with the early fermentation stage (Andrés-Barrao et al. 2012 Wu et al. 2017). Overall, the aforementioned studies showed that the universal stress mechanism mediated by HSPs is one of the ways by which AAB ensure smooth acetic acid fermentation (Fig. 1c).


Experimental procedures

Strains, media and culture conditions

Acetobacter pasteurianus CICIM B7003 isolated from a brewing factory (Hengshun Wantong Food Brewing Co., Ltd., Xuzhou, China) was used in this study. Escherichia coli JM109 used for general cloning was grown under routine conditions, on Luria–Bertani (LB) agar plates or in LB broth at 37°C. All the bacterial strains used in this study are shown in Table 2. The seed medium contained 10 g l -1 glucose, 10 g l -1 yeast extract and 3% (v/v) ethanol. The fermentation medium contained 10 g l -1 glucose, 10 g l -1 yeast extract, 0.6 g l -1 KH2PO4, 0.4 g l -1 MgSO4 and 4% (v/v) ethanol. When required, kanamycin (50 μg ml -1 for E. coli or 25 μg ml -1 for A. pasteurianus) was added to the culture medium. Cells from cryovials were incubated in 50 ml of seed medium in 250 ml Erlenmeyer flasks, and they were cultured at 30 °C for 24 h at 170 rpm. Fermentations were performed in fermentation medium at 30 °C at 220 rpm. Different initial concentrations of acetic acid were added to fermentation medium for detection of growth and production in A. pasteurianus and mutations.

Name Description Reference or source
Strains
A. pasteurianus CICIM B7003 Acetic acid production strain Lab stock
E. coli JM109 endA1, recA1, gyrA96, thi, hsdR17 (rk – , mk + ), relA1, supE44, Δ(lac-proAB), [F´ traD36, proAB, laqIqZΔM15].. Sangon Biotech
Plasmids
pBBR1MCS-2 A broad-host vector, Kn R Wang, et al. ( 2016 )
pT-adhA Plasmid pBBR1MCS-2 containing Ptuf-adhA from A. pasteurianus This study
pT-aldh Plasmid pBBR1MCS-2 containing Ptuf-aldh from A. pasteurianus This study
pT-aal Plasmid pBBR1MCS-2 containing Ptuf-adhA and Ptuf-aldh from A. pasteurianus This study
pT-pqqAB Plasmid pBBR1MCS-2 containing Ptuf-pqqAB from A. pasteurianus This study
pT-pqqABCDE Plasmid pBBR1MCS-2 containing Ptuf-pqqABCDE from A. pasteurianus This study
pT-adhA-pqqAB Plasmid pBBR1MCS-2 containing Ptuf-adhA and Ptuf-pqqAB from A. pasteurianus This study
pT-adhA-pqqABCDE Plasmid pBBR1MCS-2 containing Ptuf-adhA and Ptuf-pqqABCDE from A. pasteurianus This study
pT-aldh-pqqAB Plasmid pBBR1MCS-2 containing Ptuf-aldh and Ptuf-pqqAB from A. pasteurianus This study
pT-aldh-pqqABCDE Plasmid pBBR1MCS-2 containing Ptuf-aldh and Ptuf-pqqABCDE from A. pasteurianus This study
pT-aal-pqqAB Plasmid pBBR1MCS-2 containing Ptuf-adhA, Ptuf-aldh and Ptuf-pqqAB from A. pasteurianus This study
pT-aal-pqqABCDE Plasmid pBBR1MCS-2 containing Ptuf-adhA, Ptuf-aldh and Ptuf-pqqABCDE from A. pasteurianus This study

Plasmid construction

All the plasmids used in this study are listed in Table 2. Plasmid construction and DNA manipulations were performed by following standard molecular biology techniques. All the primers used for PCR amplification are listed in Supplementary Table S1. Schematic diagrams of genetic constructs containing the enzyme genes from acetic acid biosynthesis pathway, PQQ biosynthetic genes and their various combinations are shown in Fig. 1.

The open reading frames (ORFs) of adhA, aldh and promoter of elongation factor TU (Gene ID: 8435080) as well as the pqqAB and pqqABCDE genes were amplified separately using genomic DNA of A. pasteurianus. The promoter of elongation factor TU was ligated with different adhA, aldh, pqqAB and pqqABCDE genes using SOE-PCR. Subsequently, the resulting fragments Ptuf-adhA and Ptuf-aldh were inserted into KpnI-BamHI sites of the pBBR1MCS-2 plasmid using In-Fusion Cloning, resulting in plasmids pT-adhA, pT-aldh and pT-aal. The fragments Ptuf-pqqAB and Ptuf-pqqABCDE were digested and inserted at SpeI-PvuI sites of the pBBR1MCS-2 plasmid to produce pT-pqqAB and pT-ABCDE, and they were separately inserted into pT-adhA, pT-aldh or pT-aal plasmids, generating six plasmids with different gene combinations (listed in Table 2). All the constructs were transformed into A. pasteurianus by electroporation (Zhang, et al., 2010 ).

Analytical methods

The cell growth was monitored based on OD value at 600 nm using an EnSpire 2300 microplate reader (PerkinElmer, Waltham, MA, USA). The standard curve between OD600 and number of living bacteria (N) was obtained in A. pasteurianus and described in Fig. S1 (N = 10 3.1666*OD+7.0226 ). The growth rates were determined from exponential growth phase using the three parameters in the fit of ln(N/N0) vs time curves proposed in Bershtein, et al. ( 2015 ). The relative fitness value (W) was calculated by finding ratio of the growth rate (mutant: ancestor) (Liu, et al., 2019 ). The total acid content was measured by titrating against 0.1 M NaOH with phenolphthalein as the pH indicator. The concentration of ethanol was determined by Hitachi HPLC system with an Hi-Plex Ligand Exchange column (Agilent, 7.7 × 300 mm, 8 µm particle size). In this study, all experiments were performed in triplicate. The results were expressed as average values with a standard error.

Measurement of PQQ

The PQQ concentration was measured using crude enzymes from E. coli/pET-28a-gcd containing apo-glucose dehydrogenase with some modifications described in Wang, et al. ( 2016 ). In short, 500 μl of enzyme solution containing 250 μL of crude enzyme (approximately 0.4 mg protein), 250 μl of sample or a specific amount of PQQ standard and 10 mM MgSO4 in 50 mM phosphate buffer (pH 7.0) was incubated at 30 °C for 30 min. The reaction mixture was prepared by incubating 100 μl of enzyme solution, 0.20 M substrate glucose, 0.67 mM phenazine methosulfate (PMS) and 0.1 mM 2,6-dichlorophenolindophenol (DCIP) in 1.0 ml of phosphate buffer pH 7.0 at 30 °C for 5 min. The absorbance changes in the reaction mixture were measured at 600 nm once the D-glucose was added. The protein concentrations were measured using a Bradford Protein Assay kit (purchased from Sangon Biotech, Shanghai, China).

Semi-continuous fermentation

Semi-continuous acetic acid fermentation was performed in a 7.5 l fermentor like our previous work (Qi, et al., 2014a ). For starting-up process, 3.16 l fermentation medium containing 10 g l -1 acetic acid was poured into fermentor and mixed adequately with 0.3 l seeds. Aeration rate was set at 0.865 l min -1 (0.25 vvm). When the residual ethanol concentration was below 5 g l -1 , 0.54 l fermentation medium with 260 g l -1 ethanol was supplemented into fermentor to continue the starting-up process. Simultaneously, aeration rate was set at 1.2 l min -1 (0.3 vvm). Temperature was set at 30 °C for whole process. Starting-up process was completed when the acetic acid content increased to about 70 g l -1 with less than 5 g l -1 residual ethanol. Subsequently, a new repeated batch was operated with discharging 43% (v/v) of total working volume (4 L) and then feeding the same volume of fresh fermentation medium containing 81.4 g l -1 ethanol. Then, an acetification process was occurred as the previous one.


Introduction

Bacillus cereus is a common human pathogen that can cause two distinct types of food-borne diseases and other types of infection ( Kotiranta et al., 2000 ). Upon ingestion, diarrhoeic strains can produce enterotoxins, such as haemolysin BL, cytotoxin K and non-haemolytic enterotoxin ( Schoeni and Wong, 2005 ), causing abdominal pain and watery diarrhoea ( Stenfors Arnesen et al., 2008 ). The other type of food-borne illness involves intoxication caused by the emetic toxin cereulide produced by some B. cereus strains ( Ehling-Schulz et al., 2004 ). Cereulide is pre-formed in food and because it remains stable upon heat and acid exposures, the toxin is still active after cooking and stomach transit ( Kramer and Gilbert, 1989 ). Upon ingestion of cereulide typical symptoms may occur within 1–6 h that resemble Staphylococcus aureus intoxication ( Le Loir et al., 2003 ), including nausea, vomiting and general malaise. The symptoms are generally mild however, in rare cases liver failure has been noted resulting in fatalities ( Mahler et al., 1997 Dierick et al., 2005 ). Besides being an important food-borne pathogen, B. cereus is also a notorious food spoilage organism. Food spoilage is caused by growth of unwanted bacteria in food and causes enormous expenses for food industry ( Gram et al., 2002 ). Bacillus cereus mainly causes spoilage of milk and dairy products, because it is able to form endospores. These spores are survival vehicles formed upon nutrient shortage and are metabolically inactive ( de Vries, 2006 ). Spores are extremely resistant to stress conditions, such as radiation, high temperature, freezing, drying and acid conditions ( Setlow, 2006 ).

Spores and vegetative cells of B. cereus can be found in a wide range of environments (Fig. 1), such as soil ( von Stetten et al., 1999 Vilain et al., 2006 ), plant rhizosphere ( Berg et al., 2005 ) and various foods ( Choma et al., 2000 Rosenquist et al., 2005 ). Bacillus cereus can also be isolated from faeces of healthy adults ( Ghosh, 1978 ), suggesting that B. cereus can be part of the microbiota found in the human gastrointestinal tract. The human stomach and small intestine are acidic environments that have to be overcome by spores and/or vegetative cells to become infectious. Outside the human host, B. cereus may also be frequently exposed to acidic conditions including a vast array of foods at low pH, where in specific cases organic acids have been added as preservatives ( Keijser et al., 2007 ). Additionally, the natural reservoir of the soil saprophyte B. cereus may also be acidic upon the exudation of protons and organic acids in the plant rhizosphere ( Neumann and Martinoia, 2002 ). The antimicrobial activity of organic acids is pH-dependent with the maximum effect occurring at low pH values. At these low pH values organic acids are in undissociated states. Because undissociated acid molecules are uncharged and lipophilic, they will penetrate plasma membranes and thus enter cells. Theoretically, the higher-pH environment of the cell's cytoplasm promotes the rapid dissociation of acid molecules into charged protons and anions. These charged molecules cannot subsequently diffuse back across the plasma membrane. Thus, a permeant organic acid stresses the cell by importing protons, depressingcytoplasmic pH, and by concentrating the organic anion within the cytoplasm in proportion to the transmembrane pH difference ( Brul and Coote, 1999 ). These effects may be counteracted by the cell at the expensive ATP when it tries to extrude protons or metabolize undissociated organic acid molecules ( Mols et al., 2010b ). Apparently, coping with acid conditions is a determining factor in B. cereus' successful colonization of different niches.

Transmission routes of the food-borne human pathogen Bacillus cereus, with a variety of niches indicated from which vegetative cells and/or spores can be isolated ( Mols, 2009 ).

Acid stress responses of Gram-negative organisms, such as Escherichia coli and Salmonella Typhimurium ( Richardson et al., 2001 ), and in a select number of Gram-positive bacteria, such as lactic acid bacteria and Listeria monocytogenes ( van de Guchte et al., 2002 Cotter and Hill, 2003 Ryan et al., 2009 ) have been reviewed. These reviews highlight the importance of proton pumps, i.e. F1F0-ATPase, transcriptional regulators, such as RpoS (Gram-negatives) and σ B (Gram-positives), proteins involved in protection of macromolecules, such as DnaK and GroESL, and enzymes that produce alkaline compounds, such as the ammonium-forming enzymes urease and arginine deiminase. Until recently, no detailed information was available on the acid stress responses of B. cereus. Fluorescence techniques, physiological studies and transcriptome analyses elucidated acid stress responses of vegetative cells and germinating spores of B. cereus, including novel observations such as the formation of reactive oxygen species (ROS) and the induction of a secondary oxidative stress response ( Thomassin et al., 2006 Mols et al., 2009 2010a , b den Besten et al., 2010 Biesta-Peters et al., 2010a , b van Melis et al., 2011a ). The aim of this minireview is to provide an overview in the physiological responses, possible acid-inflicted damage and protective mechanisms displayed by B. cereus upon exposure to acid conditions.


Fermentation Vs Respiration : Definition, Types and Differences

The term ‘ferment’ is derived from the Latin word ‘fervere’ meaning "to boil." In the late 14th century, alchemists described fermentation process and it became the subject of scientific investigation in the 16th century. In the 1860s, Louis Pasteur studied the fermentation process. In 1897, German chemist Eduard Buechner first used fermentation process scientifically and fermented a sugar solution. His experiment is considered the beginning of the science of biochemistry which earned him the Nobel Prize in chemistry in 1907. Hence, the study of fermentation is known as Zymology. To make different industrial products such as wine, cheese, beer yogurt, and other products manufacturers apply the fermentation process.

Fermentation is the metabolic process by which organic molecules such as glucose, starch or sugar are converted by micro-organisms into acids, gases, or alcohol under anaerobic condition. To get energy yeast performs fermentation by converting sugar into alcohol while bacteria convert carbohydrates into lactic acid through the fermentation process. Generally, bacteria and yeast need an oxygen-free environment to live. Many beverage and food industries use the fermentation process to make the conversion of sugars into ethanol. In this case, ethanol is used to produce alcoholic beverages by using yeast which releases CO2.

Types of Fermentation

There are many types of the fermentation process. Among them, the most common fermentation processes are ethanol and lactic acid fermentation. People produce commercial foods such as beer and bread by using an ethanol fermentation process. Lactic acid fermentation is used to flavor and preserve dairy products and vegetables.

Many foods and beverages industries use the fermentation process to produce many important industrial products:


Abstract

Acetic acid bacteria (AAB) live in sugar rich environments, including food matrices, plant tissues, and the gut of sugar-feeding insects. By comparing the newly sequenced genomes of Asaia platycodi and Saccharibacter sp., symbionts of Anopheles stephensi and Apis mellifera, respectively, with those of 14 other AAB, we provide a genomic view of the evolutionary pattern of this bacterial group and clues on traits that explain the success of AAB as insect symbionts. A specific pre-adaptive trait, cytochrome bo3 ubiquinol oxidase, appears ancestral in AAB and shows a phylogeny that is congruent with that of the genomes. The functional properties of this terminal oxidase might have allowed AAB to adapt to the diverse oxygen levels of arthropod guts.



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