05: microbial metabolism ii

8/12/2019 05: Microbial Metabolism II

http://slidepdf.com/reader/full/05-microbial-metabolism-ii 1/11

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Up to this point, we’ve looked at a bacterial cell. Looked at bacterial cytology. Seen what these cells are

made of. What’s inside the bacterial cells. Ribosomes, etc., appendages, the cell membrane, the cell

wall with capsule. Spent a lot of time going over then structure of the peptidoglycan especially, that’s

important to know. Everything you can, I’d tell you about it because it’s gonna come up over and over

again in this course. And today we’re going to take a look at what goes on inside a bacterial cell.

Metabolism. Now I know you’re already well versed in this topic because you had the course in building

blocks just a little over a year ago. You’ve gone through the glycolysis, the TCA cycle, electron transport,

etc, things like that. I just want to mention because they do indeed happen in bacteria as well, with a

couple of modifications, that’s all. But just because I’m gonna review it now, doesn’t mean you don’t

have to know it. Whatever I say-, I’m trying point out some of the highlights of metabolism that you’ve

already gone through step by step. I’m not going to ask you for every step in glycolysis or the TCA cycle,

electron transport. Basically, what are they used for, what are these systems used for when bacteria

and even our cells grow. How they get food, how do they break down food, how do they get energy to

do what they have to do? So that’s metabolism. And I guess we’re already about an hour behind, but

I’ll try to finish up if I can today. If not, I have one more lecture before the first exam. And I’ll finish upthat time next week, if I don’t do it today. So uhh…

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Metabolism. Metabolism are really all of the biochemical reactions that occur inside of cells. Bacteria

(prokaryotes) and also eukaryotes (us, animals, and plants). There are two parts of metabolism.

Anabolism, which refers to the actual synthesis of the macromolecules in our cells – the proteins, the

lipids, the carbohydrates –built up of building blocks like amino acids and simple sugars, etc.,

nucleotides. That’s anabolism. Making these large structures inside of our cell. Anabolic steroids. You

take them to build yourself up, you get muscular. So those are the steps involved in biosynthesis. And

these steps to make things in cells and bacteria and in us require energy. And catabolism are the part of

the metabolism that produces energy. For the cell to use for anabolism. To produce things, tosynthesize things.

So energy requirements in general. We’ll see those in the next slide. All cells need energy. And as you

know, in metabolism, you go back and look at glycolysis and other systems that occur in the cell. Series

of oxidation-reduction reactions. So one substrate is oxidized, another is reduced, over and over again.

A whole chain of reactions occur as you’ll see in glycolysis briefly, coming up. So oxidation-reduction

reactions occur in metabolism.

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Why do you need energy? Why do bacteria need energy? For anabolic reactions, for transport, that is to

get things from outside the cell, foods from outside in the cellular environment to the interior of the cell,

in the cytoplasm, where they can use the food that’s been transported for growth and energy. Motility

use flagella. That takes energy. Cell division itself, where one cell grows and divides into two, requires

energy in the form of ATP. And maintenance, just staying alive. Even when we’re asleep at night, we’’re

in bed. To stay alive, we still are undergoing metabolism. We don’t just give up. We’re on a very low

rate of metabolism, but bacteria, too. To maintain, to stay alive, when we’re not growing that well.

Need energy just for that.

How do the cells derive energy? Oxidation-reduction reactions. One substrate is oxidized, another is

reduced. Oxidation of substrates (food, metabolites) is really hydrolysis of them. It’s catabolism,



oxidation, breakdown of foods to produce energy. The energy is produced and trapped. And as you

know, the storage compound for cellular energy is ATP. So these things, like bacteria digest glucose and

other sugars. They’re fiddled(?) energy. They have internal energy in them, but you have to break them

down. You have to hydrolyze or catabolize glucose and other sugars to produce energy. And as you

break them down step by step in glycolysis and the TCA cycle, electron transport, that’s how you get

energy, ATP. Oxidation reactions, catabolic reactions.

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This is just an overview of what we’re go through very briefly, once again. Just a review of the

importance of different steps involved in catabolism. We have the proteins, sugars, and lipids can be

broken down. Here’s a bacterial cell growing in a very rich environment of large proteins and

polysaccharides and other things it cannot take inside. They’re just too large. So first they have to

degrade them into amino acids, simple sugars, and then take them inside. Glycerol. Then many are

converted to glucose. And this pathway here, when cells break down glucose to produce pyruvate, is of

course glycolysis right here. You’re familiar with glycolysis. The breaking down of glucose. That’s the

typical example in all courses in biochemistry and building blocks. How cells begin to derive energy.

We’re gonna look a little bit at that glycolysis. You can see the glucose, one molecule of glucose. Howmany carbons are in glucose? 6. How many are in pyruvate? 3. Right. So you’re breaking down

glucose to pyruvate in glycolysis. Another name for it is the Embden-Meyerhof pathway, remember

they use that. You get two molecules of pyruvate. two 3-carbon compounds, pyruvate. And this

reaction, glycolysis, can occur anaerobically. You’ll learn more about what that means in about twenty

minutes or so. Anaerobic, it does not require oxygen. It does not require molecular oxygen. Right now,

we require, we need oxygen to grow. Right now, we’re breathing air. And there’s about 20% free

oxygen, O2. We need that, we wouldn’t survive without oxygen. But this reaction can go on inside a cell.

This pathway, glycolysis, doesn’t need oxygen. Later on, we’ll see oxygen is important, but glycolysis

itself, can occur under anaerobic conditions. It can occur in aerobic as well, but we’ll talk about both of

those later on. But the key here is glycolysis can occur under anaerobic conditions. You don’t need

oxygen.

And then the pyruvate, once you get to pyruvate in cells, there are many different, what they call fates,

for pyruvate. Pyruvate can go off in many different directions. Many, many things can be done by cells

(and bacteria cells in particular) to pyruvate. And one of the most important of course is using pyruvate

to go into the TCA cycle, the Krebs cycle. Combines with acetyl-CoA to go into another Krebs. And this

occurs only aerobically. Aerobically. So, we’ll go back to point these things later on as well. So TCA

cycle cannot occur under anaerobic conditions. Only with air, oxygen, free molecule oxygen. And then

from there, the TCA cycle, the molecules are broken up further to get an electron transport chain to get

ATP. And that’s where most of the energy-. ATP is derived in cells by electron transport. So that’s the

overview, I think you’re familiar with that. Glycolysis. Glucose to pyruvate. pyruvate, we’ll see, can go

into the TCA/Krebs cycle, or it can do many other things. I’ll point out some of them. And then from the

TCA cycle going around, around, and around, and then being changed, molecules being produced,

sending them into the electron transport chain, where you get energy production. So here is how you

get the ATP. And they say, it used to always be the number of ATPs that you can get under respiration

such as shown here, these pathways, What’s the total number? Anybody remember? 30-something? 38,

right. But now some people say 36 or 38 ATP. That’s a tremendous amount of energy. 38 ATPs (are

shown below right there) can be derived from the breakdown of glucose. That’s sugar. Glucose itself.

Reserve of energy. You break it down. You break it down, step by step. Energy is released. Finally the

energy is shuttled through the electron transport chain. ATPs are produced, 36 to 38 ATPs. These



catabolic pathways are breaking down stuff to produce energy for greater use in growing, the anabolic

steps.

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Glycolysis. We’re going to look at ATP yield. Glycolysis we’ll see the ATP is produced. But if you recall,

the ATP produced in glycolysis is very minimal. Not many at all. But when you produce ATP, during the

glycolysis pathway, it’s called substrate level phosphorylation. And we’ll also look at the important of

molecule NAD. So we’re not gonna go through every step, but look at the what’s happening with ATP

and NAD in this reaction, glycolysis.

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So here you see glycolysis, two parts here. Glycolysis, so part one, part two, I guess. The 6-carbon

compound, glucose. A series of hydrolytic or catabolic reactions occur. One molecule is oxidized,

another reduced. Oxidation-reduction, redox reaction, over and over again. Then the 6-carboncompound, fructose, as you recall, broken down into two 3-carbon compounds, and the pathway

continues to get pyruvic acid. So pyruvic acid is what is called the end point or the end product of

glycolysis.

Let’s look at the ATP produced and used up in glycolysis. To begin glycolysis, remember one of the

reasons you were doing this, having this pathway is to produce ATP. But initially, to begin glycolysis, as

you recall, you need ATP. Kind of jumpstart the pathway. You use ATP here converted into ADP when

you use it. And here’s another step here where you have to use ATP. So to begin glycolysis, you need

two molecules of ATP.

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didn’t plug it in.

[computer back on]

Okay, saved by Dr. Li, thank you.

So here we have glycolysis, and initially you use two ATP. Break it down. But look at that here. Once

it’s broken down into 3-carbon compounds. Remember it sort of splits – these pathways going in twice

here. Once you get to fructose-6-phosphate, it’s broken down two 3-carbon compounds. You get ATP

produced here, and ATP produced here. I’m not even gonna ask you where the steps are involved in

ATP production, but you get ATP produced. But since this is going on twice, you get two ATP on one side

and two ATP on the other. You produce four ATP in glycolysis. So overall in glycolysis, you use up two

ATP to start it. You produce four ATP. The net yield of ATP in glycolysis is only two. Not a lot of energy

in glycolysis. This has to take place, this reaction, but only two ATP of the 36 or 38 we’re gonna see can

be formed in these pathways.

One other thing to look at here is the use of this molecule in NAD. Here you can see in glycolysis, it is

reduced from NAD to the reduced form. NAD is used up. I mentioned that, that’s critical to know, too.

Because NAD is critical for glycolysis. It’s necessary. It’s used up as is shown here. It’s reduced, but it is,

we’ll see, recycled. It is recycled. So it’ll kind of act like an enzyme, because it’s used over and over



again. So we’re gonna see later on where this NAD, this oxidized NAD, is reduced… is regenerated, is

reproduced, so it can be used over and over again in glycolysis. So two ATP net yield. NAD reduced, but

has to be recycled, reused later on, reproduced as we will see. And further metabolism.

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Here’s the TCA cycle. Remember, it’s called an amphibolic cycle. Put that word down. This is critical.

Dentists always say when they think back upon their basic science, years in dental school. “Oh boy, I

remember the TCA cycle, the Krebs cycle. I still use that every day of my life.” That seems to be the big

whipping boy. I know, but it’s critically important. I mean, you don’t have to know every step of it, but

this is what keeps us alive, the TCA cycle. It’s involved in both catabolism and anabolism. It’s involved in

further energy production in catabolic reactions, as well as producing these compounds that are used by

cells to make other things. So it’s involved in both types of central reactions that occur in metabolism.

Catabolism and anabolism. So that’s basically what it’s used for. And here we see one fate of pyruvate

is to enter the TCA cycle. Forget about all of these, but you see two hydrogen atoms come off here,

here, here, and all these hydrogen atoms are gonna go into the electron transport chain. And that’s

where most of the ATPs are gonna be formed. So over and over again, energy is going to be produced

because of these hydrogen atoms being produced here. And also you have all of these molecules beingformed, too. They are sometimes withdrawn from the cycle, right? And they are used for the building

blocks for our proteins and our nucleotides and our polysaccharides. So that would be energy

production. Also, finishing the building blocks of macromolecules as they’re withdrawn from the TCA

cycle. So what happens to these hydrogen atoms? As you know…

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Hydrogen atoms are composed of a proton, positive charge, and an electron, negative charge. And one

easy way to remember this, in case you can’t remember it off of your head. A story about these two

hydrogen atoms that were walking along the street in the opposite direction in the sidewalk. And they

hadn’t seen each other for quite a while. And they bump into each other on the street, these two

hydrogen atoms. And the first one says to the second one, “How are you doing?” The second one says,“Well, not too well. I think I’ve lost my electron.” And the first one says, “Are you sure?” The second

one says, “I’m positive.” *chuckles+. That’s how I remember it. Electrons, protons. Hydrogen atoms.

So here we have these hydrogen atoms, and as they-. And then the electrons and protons are separated

from each other in this pathway here. Electron transport. The electron are transported. The hydrogens,

the protons, are sort of put outside of the bacterial cell. Just like in mitochondria, the protons are put

outside that inner membrane, and they’re kind of dying to get back in, trying to force themselves back in.

And the electrons are inside being transported along the electron transport chains by the cytochromes,

the dehydrogenases, and other components of electron transport. They’re being transported while the

protons are kind of hanging around outside. The cell in the case of bacteria outside the cell membrane,

trying to get back in, neutralize that electron charge. So electrons are being transported down the

electron transport chain. And once again, [mumbles], but you can see, ATPs are being formed. Up to 36,

38 ATPs when all of this goes on inside a cell that’s growing aerobically. So finally you get the third ATP,

in addition to the others in glycolysis and over and over again, up to 36 or 38. And finally, why do we

need oxygen to survive? I remember reading about bacteria when I took a micro course. I remember

reading about these bacteria called anaerobes that can grow without oxygen, and I thought, that’s

impossible! How can you grow without oxygen, free molecular oxygen? I mean, all cells need oxygen

for their carbohydrates and they’re carboxyl groups, but as far as free molecular O2, we need it. And

why do we need it? It’s because of this last step of electron transport. That’s why we simple need



oxygen among many other [things] to survive, really. Oxygen. And I wanna point this out in a better

slide that shows this terminal step of electron transport. So you go to glycolysis, you go to the TCA, the

electron transport, and finally after electron transport is over, what do you get? Water, H2O. That’s the

end product of all those metabolic pathways that occur in bacterial cells that are growing aerobically.

And oxygen we’ll see play an important role. And finally saying this is enough. We’re done with this

pathway now, these hosts of other sequences of different pathways are now over.

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Okay, here it is, here again. And this is called respiration. Respiration, where phosphorylation occurs.

Where ATP is produced, that’s called respiration. It can be aerobic or anaerobic, respiration, ATP

production. Aerobic respiration here. And here’s that last step once again in electron transport.

Electron reactions. Finally, those electrons are being transported and those protons that were outside

the membrane just dying to get in, from the hydrogen atoms that began the whole process recombine.

Get together with oxygen, molecular oxygen to form water. So another way to refer to this process in

aerobic respiration where ATP is formed, the last step where water is formed, as you say, the oxygen is

the final electron acceptor. In aerobic respiration, where oxygen is used. Oxygen itself is the final

electron acceptor, by definition, aerobic respiration. And that’s why it finally picks of the electrons. Itpicks up the protons, too, but-… you can say it’s the final proton acceptor, too, but by convention, we

say oxygen in aerobic respiration is the final electron acceptor. Form water, water is the end product.

And then you’re done.

In anaerobic respiration, we’ll see these are bacteria in the absence of oxygen. They lack the

cytochromes required to use oxygen as the final electron acceptor. So anaerobic bacteria, as the name

indicates, these cannot use oxygen. Another fact, to many anaerobes, oxygen is a poison. And we’ll see

where that can occur, too. They can’t deal with oxygen. So bacteria that grow anaerobically use

something-. They still have to go through glycolysis. They don’t go through TCA cycle, but they go

through glycolysis, they go through electron transport. But at the end, instead of using oxygen, when

they’re anaerobic bacteria growing without air, they will use other electron acceptors, such as nitrate,sulfate, or carbonate. So by definition aerobic respiration use oxygen as the final electron acceptor and

anaerobic respiration – and a lot of bacteria we’re gonna talk about are strict anaerobes that can occur

severe oral infections like periodontal disease – the final electron acceptor in their respiration is either

nitrate, sulfate, or carbonate. NOT oxygen. So another way to put it, the final electron acceptor in

anaerobic respiration is an inorganic compound other than oxygen. Because oxygen is an inorganic

compound, right? But these are inorganic compounds that acceptor electrons and they make

respiration. That’s why these bacteria can grow anaerobically, they don’t need oxygen. Oxygen can

harm them. They use other things. They have to carry out glycolysis and electron transport. A different

electron transport chain in anaerobes, but they use it to derive energy in their electron transport chains

as well. In anaerobes.

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So you can classify bacteria by the effect of oxygen on their growth. Obligate aerobes means some

bacteria will only grow in the presence of oxygen, in air, O2. Air and oxygen, we use them

interchangeably now. Obligate aerobes.

Obligate anaerobes, will not grow or will not grow well – there’s a whole gradient of different strict

aerobes to strict anaerobes of bacteria. But anaerobes mean they do not grow in the presence of

oxygen. As a matter of fact, oxygen is a poison to many strict anaerobes. Obligate or strict anaerobes.

We’re gonna talk about the importance of these two enzymes, and which should determine whether or



not a bacterial cell is an aerobe or an anaerobe. These two enzymes we’ll see in the next slide are

critically important in that role.

There are bacteria known as facultative anaerobes. They can grow in the present or absence of oxygen,

of air. Most bacteria are really facultative anaerobes. Can grow with or without air.

Microaerophilic bacteria are those that grow with a very small percentage of oxygen. Right now, what is

the percentage (I think I asked this before), the percentage of oxygen we’re breathing? It’s about 20%,

right? Okay and in our mouth, it’s maybe 10%, 12%. And then farther back in the mouth and down

there in the gingiva, the gingival sulcus, between the teeth and the gums, there may not be any free

oxygen at all. That’s a good anaerobic environment. So some-. 20% is optimal for most bacteria if

they’re aerobes. These *Microaerophilic bacteria+ grow to about 5% oxygen best. They thrive, they

grow much better under 5%. We have special incubators we’ll see that enable them to grow. You can’t

grow them on a Petri plate and stick them in a regular incubator and have them grow well. You have to

reduce the oxygen content from about 20% to 5%. Microaerophilic.

And some bacteria, we characterize them as capnophilic. Whenever you see “-philic” of course that

always means “love” – they love something. And these are bacteria that love “capno-“ – they love

carbon dioxide. This is another thing. Many bacteria are stimulated in their growth. They grow much

better when you enrich their environment with carbon dioxide. And we also have incubators that dothat as well. You put the plate in the incubator, you have this tank of carbon dioxide, a tube lead from

the tank of CO2 into the incubator, and CO2 builds up inside, interior of the incubator to about 5% as well.

5% CO2 and they grow much better than they would in the absence of CO2, these capnophilic bacteria.

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Here, an obligate aerobe would be Bacillus, that genus. Obligate anaerobe would be Clostridium.

Examples of facultative anaerobes are Streptococci that cause caries, E. coli , and most bacteria. So

microaerophiles we’ll talk about when we get to infectious diseases, as well as capnophilic bacteria. But

majority of bacteria in the oral cavity are either obligate anaerobes, not Clostridium necessarily, but

those down in the gingival sulcus. And we’ll talk about the cause of periodontal disease or facultative

anaerobes that can grow with or without air. Streptococci , you’ll learn an awful lot about that particulargroup of bacteria. E. coli as well.

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So what is it about oxygen that kills-, as I said oxygen can act as kind of a poison to anaerobes. Well

sometimes, well always, really, in metabolic reactions, imagine a bacterial cell growing and doubling

every 20 minutes. There must be a ferocious amount of activity going on inside that cytosol of the

bacterium. Within 20 minutes, they can lose all the cell wall, and the flagella, the DNA and RNA. Every

now and then, some things in metabolism that are harmful for cells unless they’re gotten rid of

instantaneously within nanoseconds. And some of these things that are dangerous that are toxic to

bacteria are reductions, molecular oxygen being reduced. Here’s oxygen. Here’s what we call the

superoxide radical. And here, the negative charge. Hydrogen peroxide. And even the hydroxyl anion.

One, two, three. These three things are very dangerous to bacteria. If they survive even a second or

longer, they’re gonna do some damage to the components of our cells, like our DNA, RNA, proteins, etc.

So you have to immediately get rid of these dangerous reduced compounds of oxygen. Superoxide

anion, hydrogen peroxide (which is used as you know a disinfectant and antiseptic), and hydroxyl ion.

So that’s bad. Why do these things that are dangerous and they are produced in both aerobes and

anaerobes, so why is it that aerobes can exist when they’re produced, whereas anaerobes cannot exist

and survive when these compounds here are produced.



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Well, here’s what happens. Why are anaerobes killed by exposure to oxygen? Here’s a superoxide

radical, and here’s H2O2, and also OH, the hydroxyl on it are oxidizing agents. Oxidizing agents we’re

always told… we have to take vitamins and things to get rid of our oxidizing agents so they don’t harm

ourselves. The superoxide radical, hydrogen peroxide, and also the hydroxyl ions, should have that here,

are oxidizing agents. Generated during metabolism, split nanosecond, they’re gone.

Aerobes, but not anaerobes, contain these two enzymes that help aerobes survive. Superoxide

dismutase and catalase. Aerobes have these two enzymes, anaerobes do not, they lack them. Aerobes

can do, when they grow, is what they call the enzyme superoxide dismutase that mismutates, or

changes, the superoxide radical, shown here. This enzyme here, cause a disreaction when you get H2O2

plus oxygen. Now this is also dangerous, so right away, aerobes breakdown H2O2 using the enzyme

catalase to form water and oxygen. Danger of oxidizing agents to cells. So that’s what happens.

Aerobes have these important enzymes that maintain their safety and a lot of them survive, whereas

anaerobes do not, and these hydroxyl ions and superoxide radicals build up and they destroy, danger,

destroying DNA, protein, other components of the cell. And the cells will die. So anaerobes will die very

quickly in the presence of these molecules shown here because they can’t handle them, to let them

build up and these molecules shown here destroy components of the anaerobic cell.

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Okay we talked about aerobic respiration and anaerobic respiration. And there’s one other type of

pathway carried out by bacteria. And it’s very important to all of you, I think, and to all of us, really. In

addition to aerobic and anaerobic respiration, bacteria can also carry out fermentations. Fermentations.

And I’m gonna mention a couple of them. One of them, well two of them shown here are lactic acid and

alcoholic fermentation. I’m gonna show you the pathway of fermentation for the production of these

molecules, lactic acid and alcohol. Lactic acid is important because that’s the one that causes the

demineralization of our enamel. When bacteria, such as Streptococci we will discuss and others,

produce lactic acid when they’re growing and the dental plaque, this biofilm on our teeth, lactic acid

begin the demineralization, begin to dissolve our enamel. Eventually lead to holes and cavities andcaries. So that’s the result of bacterial fermentation. Lactic acid we will see. And alcoholic

fermentation is also reactions where ethyl alcohol is produced, which is very important to all of us,

particularly on the weekends when we have alcoholic beverages. But also many products that bacteria

produce in fermentation reactions are used to help identify them in the microbiology laboratory. Many,

many tests are available to identify bacteria based upon what kind of products they produce when they

ferment sugars. Fermentation, how is it defined? It’s defined as a process in which the final electron

acceptor is an organic compound. So fermentation reactions are reactions and pathways in which the

final electron acceptor is some organic molecule. Not oxygen, not nitrate or sulfate or carbonate, but

organic, a carbon-containing compound acts as the final electron acceptor in fermentations. Let’s take a

look at a couple here.

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This is well, you can see how involved this must be. Here’s what bacteria can do. Pyruvate. They get

pyruvate. Talked about all the end products of pyruvate, the end products. Some bacteria even carry

out this one, lactate. All these things can be formed. Alcohol. Others, butanediol, etc, etc. So these are

just a few of the many types of reactions can occur in bacteria. To help differentiate them from each

other. Not all carry out all these reactions, of course. But one or two, or few of them. But it’s by

determining what they produce when they change pyruvate to these different compounds. Identify

formate, identify acetate, identify butyrate. Then we can help identify bacteria. Series of reactions over



and over again, to see yes or no? Do they produce this fermentation end product or not? So

fermentation is often useful in helping identify bacteria by the end products that are formed during

bacterial fermentation.

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Here’s a very simple one that’s important to you. Pyruvate. And here we have… this is a fermentation.

This is a complete fermentation reaction. Glycolysis produce pyruvate. Here we see the pyruvate in one

simple step. Look what happens here. It’s converted from pyruvate to lactate. Lactic acid or lactate.

Lactate always the salt. This is the culprit in caries. This is what demineralizes your teeth. This is what is

produced in the dumb plaque on the surface of your teeth. So here’s pyruvate. Here’s an enzyme,

lactate dehydrogenase (E for enzyme) that immediately converts pyruvate to form – some bacteria like

the Streptococci – to lactate. That’s a fermentation. That’s called lactic acid fermentation. And that

pathway is over. Lactic acid fermentation, just one simple step. Look at also here, we see that this

fermentation pathway, this fermentation reaction, NAD is reproduced. Remember I talked about it’s

important for glycolysis to have the NAD regenerated. NADH was reduced, for glycolysis has to be

regenerated, NAD. Now this can go back into glycolysis and be used over and over again. So

fermentation pathway, one simple step. Lactic acid form. Many Streptococci and other bacteria carryout this fermentation and produce tons and tons lactate on our teeth. A lot. To demineralize them. So

that’s a fermentation.

What is the final electron acceptor? Remember my definition of fermentation? In this pathway, which

compound shown here is the final electron acceptor? Gotta be one of these three, pyruvate, NADH, or

lactate. Actually it is… Where is the final electron in this reaction? It’s in here. We wanna oxidize it. So

the final electron acceptor, an organic compound, that picks up that final electron is pyruvate. So here,

in lactic acid fermentation, pyruvic acid is the final electron acceptor. It picks up the electron and is

converted to lactate.

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How about this one. Here’s another fermentation where ethyl alcohol is produced. And actually thisoccurs more in yeast and fungi. A few bacteria can carry out this fermentation, but as far as in the world

of microbes, mainly it’s the yeast that ferment sugars, grapes, sugars, and other sugars to form alcohol.

It’s a fermentation reaction. We need alcohol in our practices as well. We use alcohol for disinfection.

So it’s not just for drinking. Here pyruvate formed. The fate of pyruvate here, CO2 lost, acetaldehyde.

Here’s our friend, NADH, that was produced in glycolysis, has to be oxidized to NAD, and you form ethyl

alcohol, ethanol. Pyruvate decarboxylase is the enzyme involved here. What is the final electron

acceptor in alcoholic fermentation? What picks of the electron from NADH? Acetaldehyde, an organic

compound. So fermentation reactions in which the final electron acceptor is an organic compound.

Previously with pyruvate, with lactate fermentation. In alcohol fermentation, acetaldehyde picks it up.

Acetaldehyde is the final electron acceptor. Fermentation. Now, you get NAD regenerated in these

fermentation. That’s really great for the glycolysis to go on, it’s essential. But bacteria that carry out

fermentation don’t really get much energy at all in the form of ATP. The only ATP they get is formed in

glycolysis. So remember glycolysis in bacteria that ferment, glucose, pyruvate is formed, and then they

convert it to ethanol, in this case for example. But no more ATP is formed. So they don’t grow very well

at all in lab. They don’t have robust growth because they don’t produce much energy, but that net yield

from glycolysis helps them survive. And glycolysis can also occur anaerobically. It can occur under

anaerobic conditions, even though we see the final electron acceptor is an organic compound.

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Here we have it again. Okay, I guess I was gonna ask you the same questions. Two enzymes involved.

This is a fermentation pathway. So we have aerobic respiration, anaerobic respiration, we have

fermentation. Those are the big three that occur in bacteria. And once again, many important

compounds are formed. We just mentioned a couple of them here. The lactate, the ethanol in

fermentations. And many, many other compounds are formed to help identify the bacteria.

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Here’s an example of that. Here pyruvic acid. You can see some bacteria and do an awful lot of things,

form an awful lot of different end products. Here it’s called mixed acid fermentation. So some bacteria,

pyruvate, they can do three or four or five different things with it. Form different acids, even. And so

it’s called mixed acid, many different acids may be the end products, such as acetic acid, succinic acid.

And some even produce gases such as hydrogen and CO2. So let’s talk now how we can help

fermentations identify bacteria. Here we have a test tube. This tube here if you worked in lab, you

know what it is. It’s a glass inverted, a little tube inside the broth in this test tube. And you put that

tube in and some of the media goes in there, some of the broth goes in there. And when you take this

tube and sterilize it and bring it back, this whole tube that’s upside-down will open here at the bottom,

is completely filled with the broth and has this particular color. So this has a little gas already. Then,here we have three different tubes. In this case here, I would say… I don’t know if they look like that…

Initially, they all look the same. Initially, these three different tubes were purple. Take my word for it.

You inoculate some bacteria into this tube that do not carry out fermentation at all. The color of the

tube stayed the same, nothing happened. No acids produced, no gas. Whatever bacteria were

inoculated in that tube were not fermenters. In the middle one here, we see that indeed they did

ferment whatever sugar was in the test tube. The Durham fermentation test tube upside-down. And

when they did that, it’s acid, because the color of the media changed from purple to yellow as a result of

acid production, the pH went way down. You can see because of all the acid being formed. And also

some gas was produced. So H2 and CO2 were produced. Some bacteria can do this, others cannot. And

the other one on the left, only acid was produced, no gas. So just to point out the different usages offermentation test to, you know, say yes gas, yes no gas, yes gas and acid, or no fermentation at all. And

those are properties that help you pinpoint a microbial genus, species, and even we’ll see later, even

more accurately stereotype that are-, as well.

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And here’s one I want to mention only because it’s commonly done in the lab, called butylene glycol

fermentation. I don’t want to mention, once again, the whole pathway, but look at this compound here,

Acetoin. You’ve probably never heard of this anywhere else, I don’t know. It’s a very rare compound,

but some bacteria when they breakdown pyruvic acid, they form butylene glycol, and in the process

they form the compound called acetoin. Forget about the structure of it, but the thing is, it’s a

compound that’s produced in fermentation by some bacteria that’s easily identifiable. A very simple

biochemical test. You have the bacteria growing in a broth test tube overnight. The next morning, you

say, “I wonder if acetoin was produced overnight or not?” You add a particular reagent, and if it did

produce acetoin, the color would change to red. Doesn’t matter *the color+, but it’s a color test. And if

those bacteria didn’t produce acetoin, did not carry out this fermentation pathway, the red color would

not appear. So once again, another fermentation case, not the end product, but the intermediate in the

pathway that’s easily identifiable by Voges-Proskauer test. And we know for example, here are two

bacteria, Enterobacter and E. coli . These are very hard to distinguish between in the lab. They look alike,

they grow alike, so many different tests that are identical to these two Gram-negative bacteria. But this



is one here that helps differentiate these two Gram-negative bacteria. They look alike, stain alike,

everything. And that’s because Enterobacter , that particular genus does produce acetoin. E. coli does

not. Very, very useful because E. coli , as we will see, can do a lot good, but also a lot of damage. It can

contaminate water, and that’s indicative of sewages. Often running the test to see whether or notE.

coli is present in what we think is contaminated water, sewage in our drinking water. Look for E. coli , if

the Voges-Proskauer test is negative, it wasn’t E. coli there, maybe the water is safe to drink. It could be

something else, but maybe it’s safe to drink, if E. coli is not there because acetoin was not found.

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This is just another slide. You can look over it after. I’ve mentioned some of these already, but just to

show you, so many different pathways of pyruvate in bacteria.

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Another way to identify bacteria. Okay, now we’ve gone through two types of respiration and

fermentation. Some other ways to classify bacteria in addition to the effect of oxygen on their growth,

aerobes, anaerobes, facultative, microaerophiles, etc… is the temperature of growth that they prefer.

Some bacteria, believe it or not, can grow very well in the cold. In the refrigerator, even. They’re calledpsychrophiles. They grow best at about, maybe, there’s a curve... maybe from 2 degrees, 4 degrees, 8

degrees. They grow very well at 4 degrees. Psychrophiles, cold. And there’s one or two bacteria we’re

gonna talk a lot later on in the other course that grow well in refrigerator and are pathogenic. And you

really have to be aware of them, because they grow better in the cold at 4 degrees centigrade, at

refrigerator temperature than they do at 37 degrees in a regular incubator. Thermophiles grow best at

above 50 centigrade. We found some even in hot springs. They can grow at incredibly temperatures.

And better than say, our body temperature. Mesophiles are the ones that grow in between these

extremes. Our body temperature is about 37 degrees centigrade. Most bacteria that inhabit us, such as

those that live inside of us, are mesophiles. They like to live at 37 degrees centigrade. Most pathogens

we’ll talk about are mesophiles. Most of them, majority. They like body temperature because once they

get inside of us, it’s the temperature they like to grow in. They like to attack our tissues, they canhydrolyze our tissues, break them apart to get food, so they can grow and the end result, they do a lot of

damage to our body and cause infections.

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ATP requirements. I guess, I don’t know. I started off doing research when I was in graduate school

with bacteria and trying to learn all about metabolism. But it always amazed me that here’s an organism,

E. coli , shown here. E. coli can grow on this very, very simple medium we prepare in the lab that Dr.

Saxena will talk about probably on Friday, called minimal media. Media, plural. It’s growth medium is

whatever we use to grow bacteria in the lab. It’s called medium. All it takes forE. coli to grow is glucose

and 5 salts. That’s incredible! Glucose, the source of carbon. No other sugar has to be added to their

growth medium for them to grow. But you have to have salts to, you know, synthesize and produce

their phosphates, sodium, chloride ions, of course, for cells to grow. So 5 salts and only one carbon

source, and from this simple medium, E. coli can produce everything it needs to grow. Its flagella, its

capsule, its peptidoglycan, its cell membrane. Just from these simple salts and one carbon source,

glucose. This E. coli that grows on this simple medium is called a prototroph. And auxotroph is a term

I’ll use later. So E. coli can grow on this simple medium, minimal medium. It’s a prototroph. Sometimes

on these slides, we find bacteria like E. coli that need one additional supplement to grow, an additional

growth factor. And that’s called an auxotroph. So an auxotroph is a bacterium that needs a growth

factor that it’s prototroph, or parent E. coli , did not need. So let’s say E. coli grows on this medium, it’s a



prototroph. If you also have to add the amino acid histidine to grow, that would be an auxotroph. You

have to add histidine to grow. And any other amino acid that would help, that they need to grow. Or

any other product. Auxotroph, something that require that the parent E. coli did not need other than

the minimal medium.

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Here’s how the energy that’s produced now in catabolism, so all that we just went through, especially

the electron transport. How is the ATP used. And from growing E. coli on that simple medium, and

following every step it uses in producing its polysaccharides and nucleic acids, every step. We know how

much energy is involved in the growth of E. coli , from a simple salt, one carbon medium, to large

population of cells. We know that the ATP that they produced, more than half is used for synthesis of

proteins. And this is true in our cells, too. We found this out initially in bacteria. How is energy used,

from catabolism, used for anabolic reactions in cells and in bacteria? And it’s the same in our cells,

roughly. 56% of ATPs produced are used for protein synthesis. Polysaccharides, 8%. Lipids, 0.3%. And

more energy is needed for RNA synthesis than for DNA synthesis. DNA synthesis does not require much

energy, not require much ATP at all. Other big uses are active transport, getting things from outside to

inside cells. And mRNA turnover 4%. So to me, that’s really incredible. And why is it? A Darwinianquestion, why is it that protein synthesis requires so much ATP? What is that step that that’s involved in

protein synthesis where ATP is used? Remember? Think about protein synthesis now. Think about that

ribosome, and there’s that string of mRNA on that ribosome. And all of a sudden, you have all the

amino acids there, lined up in a new-forming protein. And then you have tRNA coming down, right?

Transfer RNA with the new amino acid to add on to the ones that are already there to form this protein.

Every time the amino acid from the tRNA is transferred to the growing polypeptide chain, an ATP is used.

So protein synthesis is an incredibly energy demanding pathway. The production of proteins.

So I didn’t finish. I still have a couple more slides to show when I see you next week. After Dr. Saxena

sees you on Friday.

05: microbial metabolism ii

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