Anaerobe prokaryotes oxidize NADH

and other electron carriers → anaerobic

respiration (nitrate, sulfate or fumaratee

NADH (other electron carriers) is oxidize

by metabolites produced by the pathway

→ occurs in cytosol → ATP is produced

via substrate level phosphorylation

Fermentation occurs only among

prokaryotes. In yeast O2 is necessary,

unless the medium is supplemented with

sterols and unsaturated fatty acids.

Certain animal cells are capable of

fermentation, such as muscle cells &

human red blood cells

1

Fermentations are named after the

major end products they generate.

Example : yeasts carry out anas the electron acceptor) or fermentation

ethanol fermentation; muscle cellsFermentation: is a pathway in which

carry out a lactic acid fermentation.

Carbohydrates fermentation can be

grouped into six classes : lactic,

ethanol, butyric acid, mixed acid,

propionic and homoacetic

2

Electron Sinks

The fermentation pathway must

produce the electron acceptors

The electron acceptors are called as

“electron sinks”, because they

excrete the reduced products into the

medium → large quantities of

reduced organic compounds (alcohol,

organic acids and solvents,

frequently hydrogen gas)

Propionate fermentation using theacrylate pathway

Clostridium propionicum :

3 lactates → 2 propionates + 1 acetate +

1 CO2 + 1ATP

ATP is derived via substrate-levelphosphorylation, during the conversionof acetyl-P to acetate (acetate kinase).One acetate is made per 3 lactates used→ 1/3 ATP /pathway

Important!

In glycolisis, acetate is associated withthe production of 2 ATPs

Transfer of Coenzyme A from oneorganic acid compound to the others(CoA transferase)

(2H)

1

(2H) CO2

2

CoASH

3 CoASH

O

CH3 C ∼SCoAacetyl-CoA

O

CH3 C O ∼Pacetyl-P

ADP4

ATP

O

C ∼SCoAlactyl-CoA

CH3 CHOH

CoASH

6

O

C ∼SCoA

acrylyl-CoA

H2O

CH2 CH

2(H)

CH3 CHOH COOHlactate

5

O

CH3 C COOHpyruvate

CH3 COOH

acetateCH3 CH2

CH3 CH2

O

C ∼SCoA

lactyl-CoA

COOH

propionate

7X2

Enzymes :

(1) lactate dehydrogenase, (2) pyruvate-ferredoxin

oxidoreductase, (3) phosphotransacetylase, (4) acetate

kinase, (5) CoA transferase, (7) dehydrogenase. Reaction 6

is not sufficiently characterized.

3

Succinate-Propionate Pathway

Propionibacterium : Gram +, anaerobic,

nonmotile, non-sporulating, pleomorphic

rod, normal flora in the rumen of

herbivores, human skin and dairy products,

used in the making of swiss cheese

Yields more ATP than the acrylate pathway

per mole propionate formed

The Transcarboxylase Reaction Spares an ATP

Pyruvate carboxylation reaction uses pyruvate

carboxylase, CO2 and ATP

Propionibacterium : methyl-malonyl-CoA

pyruvat transcarboxylase → transfers a carboxyl

group from methylmalonyl-CoA to pyruvate

without using ATP

fumarate

4

O6(H)

1

O (2H) CO2

2

CoASH

3

CH3 C ∼SCoAacetyl-CoA

O

CH3 C O ∼Pacetyl-P

ADP4

4(H)

3CH3 CHOH COOHlactate

3CH3 C COOH

pyruvate

5

COOH

C O

CH2 oxaloacetate

2COOH

6

COOHCH3 CH2

propionate

2COOH

CH2

C HOH

COOH malate

7

2COOH

C H

C

COOH

2H2O

H

COOH

8ADP+Pi

ATP

2COOH

C H2

C H2

succinate9C H2

C H2

C ∼SCoA

Osuccinyl-CoA

2CH3

CH2

C ∼SCoA

O

2COOH

C H CH3

C ∼SCoA

propionyl-CoA

mehtyl malonyl-CoA

O

10

COOH

4(H)

2(CoAS)

ATP

CH3 COOH

acetate

enzyme:

(1) lactate dehydrogenase(a flavoprotein),

(2) pyruvate dehydrogenase(enzyme NAD+ )

(3) phosphotransacetylase,

(4) acetate kinase,

(5) mehtylmalonyl-CoA-pyruvatetranscarboxylase,

(6) malate dehydrogenase,

(7) Fumarase,

(8) fumarate reductase,

(9)CoA transferase

(10) mehtylmalonyl-CoA-racemase.

Propionibacteria can produce succinate

and propionate as an end-product of

fermentation when growing on glucose

that enter the glycolytic pathway →

having an enzyme to carboxylate C3 to

C4.

PEP is carboxylated to oxaloacetat and

then reduced to succinate. During

carboxylation, PPi is formed.

PPi is used to phosphorylate Fru-6P to

and toserineFru 1,6 bisP

phosphoserine

5

The pathway from PEP to succinate:

1. fumarate is an electron sinks

enabling NADH to be oxidized

2. fumarate reductase is a coupling

site

3. succinate can be converted to

succinyl-CoA, which is required

for the biosynthesis of

tetrapyrroles, diaminopimelic

acid, lysin, and methionine

and ButanediolMixed AcidFermentation

The enteric bacteria are facultativeanaerobes → the changes according tothe anaerobic environment:

1.The terminal reductases replace theoxidases in the electron transport chain

2.TCA becomes a reductive pathway,succinate dehydrogenase is replaced byfumarate reductase

3.Pyruvate-formate lyase is substituted forpyruvate dehydrogenase

4.Carry out a mixed acid or butanediolfermentations

6

The mixed acid and butanediol

fermentations are similar in that both

produce a mixture of organic acids,

CO2 and NADH.

Butanediol fermenters (Serratia,

Erwinia and Enterobacter) produce

large amounts of 2,3 butanediol,

acetoin, CO2 and ethanol

Mixed acid fermentation: Escherichia,

Salmonella, Shigella

Mixed acid fermentation

The reactants : PEP + CO2 or pyruvate

The products: succinate, lactate, acetate,

ethanol, formate, CO2, H2.

Formate is oxidized to CO2 and H2 by the

enzyme system formate-hidrogen liase

which contains of formate dehydrogenase

and hydrogenase. Formate dehydrogenase

oxidizes formate to CO2 and reduces

hydrogenase that will transfer electron to 2

moles of proton to form H2.

Shigella and Erwinia do not have formate-

hydrogen lyase → no gas

OAA

11 2(H)

malate

H2O12

2H

1/2 glucoseATP

1ADP

ADP + Pi

ATP

pyruvate

PEP

ADP2

ATP

3 CoASH

lactate4

Pi

CO2

10

succinate

fumarate

13 2(H)

acetyl-P

ADP

ATP

9

acetate

acetyl-CoA +

Pi 2(H)8 6

formate

CoASH

acetaldehyde

7

ethanol

H2

CO25

(1) glycolytic enzyme, (2) pyruvate kinase, (3) pyruvate-formate liase, (4) lactate

dehydrogenase, (5) formate-hidrogen lyase, (6) acetaldehyde dehydrogenase,

(7) alcohol dehydrogenase, (8) phosphotransacetylase, (9) acetate kinase,

(10) PEP carboxylase, (11) malate dehydrogenase, (12) fumarase,

(13) fumarate reductase .

7

How the pathways are balanced by

gene expression?

☼ Fnr (Fumarate nitrate reduction)

protein plays an overarching role

as a global transcriptional regulator

to ensure effective use of pathways

for fermentation and/or anaerobic

respiration

☼ Except for the presence of a

cysteine-rich N-terminal extension,

Fnr is highly similar to Crp (cyclicenzymes :

AMP receptor Protein)

8

☼ Fnr box contain a 5 bp inverted

repeat TTGAT ….. ATCAA, whereas

Crp box: TGTGA …… TCACA

☼ Fnr sense the redox condition of the

environment through the iron atom

bound to a cluster of four cysteine

residues that are highly conserved

among all bacterial Fnr protein.

☼ When the iron is in the Fe2+ state,

the protein is functionally active as a

transcriptional regulator; when the

iron oxidized to the Fe3+ state, the

protein is altered in conformation and

becomes nonfunctional.

D-Lactate formation

☺The activity of NAD+-linked D-

lactate oxidoreductase is elevated

during fermentative growth at low

pH.

Cleavage of Pyruvate

Pyruvat-formate lyase cleaves

pyruvat to acetyl CoA and formate

and encoded by the plf operon.

Transcription of this gen is activated

by Fnr in concert with ArcA

Expression of pfl under anoxic

condition can be increased further by

exogenous pyruvat

Ethanol production

Ethanol is formed by two

consecutive reductions at the

expense of two NADH molecules

The enzyme called ethanol

dehydrogenase or alcohol

dehydrogenase encoded by adhE.

Trancription is regulated by two

different mechanisms:

1. Enhancement of transcription is

associated with a high NADH/NAD+

ratio

2. repression of transcription is

exerted by the NarX/NarL system

9

Under anoxic condition, acetyl

CoA will converts to acetyl

phosphate with the help of

phosphotransacetylase. The

phosphoryl group can generate

ATP from ADP with the help of

acetyl kinase. Acetat exits the

cell, probably via a H+ - symport

system

Regulation of Formate

�������������� Format-hydrogen lyase comprising

formate dehydrogenase H (encoded

by fdhF) and hydrogenase 3

(encoded by hycE)

�������������� They convert formate to CO2 and

H2, or formate will be excreted in a

manner similar to that of acetate

�������������� An interesting feature of formate

dehydrogenase H is the presence

of a selenocystein at position 140

from the N-terminal end

�������������� The catalytic activity of Format-

hydrogen lyase requires a

molybdenum cofactor, Ni and Fe

10

�������������� The activity level of the lyase are

strongly diminished during

aerobic growth

�������������� The fdhF and hyc and hyp operon

are activated during anaerob

growth by the FhlA regulatory

protein

�������������� The FhlA protein shows partial

homology to the response

regulators of the two-component

system and respond to formate

as a signal

2H2Fdox

Fdred 4H

butiryl-CoA

Butyrate And Butanol-Aceton Fermentation

Occurs in Clostridium acetobutylicum.

Butyrate, acetate, CO2 and H2 are produced

in log phase (acidogenic phase).

Organic acids are converted to butanol,

aceton and ethanol when entering the

stationary phase (solventogenic phase).

via NADH:ferredoxin oxidoreductase

enabling the bacteria to produce more acetate

→ larger amount of ATP rather than reduce

acetyl CoA to butyrate, yet energetically

unfavorable and inhibited by the

accumulation of H2

glucoseATP

ADP

glucose-6-Ppentose

ATP

ADP

pentose-P

2NADH + 2H+

The ability to move the electron to hydrogen

ATP

ADP

Fruktosa-6-PATP

ADP

2-phosphogliceraldehyde

4ADP + 2PiNADH + H+

4ATP2NAD+ NAD+

H2

16

+

2 pyruvate17

1

2 acetyl-CoA

2CoASH

Asetoacetyl-CoA

NADH + 2H+

3NAD+

hidroxybutiryl-CoA

4H2O

Crotonil-CoA

5

ButiraldehydeNADPH + H+

15NADP+

NADH + H+

14

Butanol

11

Pi NAD+

CoASH

6ADP

butiryl-P

ATP

7butyrate

CO2aceton

8

ATP

acetate

Acetoacetateik

11

CoASH

acetaldehydeNADH + 2H+

13NAD+

ethanol

2CO2

ADP

acetyl-P9

CoASH

10

Pi

12

Fermentation without Substrate-LevelPhosphorylation

☺ The fermentation of certaincompound yields insufficient energyto synthesize ATP by substrate-levelphosphorylation.

☺ In these cases, catabolism ofcompound is linked to ion pumpsthat establish a proton motive forceor sodium motive force across thecytoplasmic membrane

☺Propionigenum modestum was firstisolated in anoxic enrichmentcultures lacking alternative electronacceptors and feed succinate aselectron donor

♣ P. modestum catabolize succinate

under strictly anoxic condition

Succinate 2- + H2O → propionate -

+ HCO3- ∆G0’ = -20,5 KJ

♣ Energy conservation is linked to the

decarboxylation of succinate by

membrane bound decarboxylase

yielding propionate

♣ This reaction releases sufficient free

energy to drive the export of a

sodium ion across the cytoplasmic

membrane

13

♣ Oxalobacter formigenes catabolizes

oxalate and produces formate

Oxalate2- + H2O → formate -

+ HCO3- ∆ Go’ = - 26,7 KJ

♣ The decarboxylation of oxalate is

exergonic and forms formate, which

is excreted from the cell