Fermentation

... when there is no external terminal electron acceptor!

Glycolysis

Substrate-level phosphorylation

2 Pyruvate
Fig.: Brock (mod.)

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The calvin cycle

glucose

ATP
reduction
NADH equivalents
respiratory chain
2 pyruvate
CO2, NADH
ATP

CO2

GTP

FADH2
NADH

The general priciple of fermentation

glucose
The problem
• Regeneration of ATP
reduction
NADH2 to NAD+ NADH equivalents
respiratory chain
2 pyruvate
CO2, NADH
ATP

CO2

GTP

FADH2
NADH
The solution
•Transfer of reduction equivalents [H] on intermediates
(e.g. pyruvate) or co-substrates

2
The general priciple of fermentation

organic substrate

degradation
ATP
intermediates

[H]

oxidised reduced
products products

Drawback
• Excretion of energy rich (reduced) substrates (e.g. ethanol)

The general priciple of fermentation

Conservation of energy not by
• chemiosmotic mechanisms (proton gradient)
but by
• Substrate-level phosphorylation
low ATP- and growth yield!

Example alcoholic fermentation: little biomass, a lot of alcohol

Bacterial fermentations are named by

their characteristical end products
alcohol (Ethanol) lactic acid
butyric acid propionic acid
mixture of different acids

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The easiest fermentative pathway

homolactic fermentation
Lactobacteriaceae
e.g.
Lactobacillus spec.

... a bit more complicated:

heterolactic fermentation

Photo: M. Dykstra, R. Barrangou,

R. Sanozky-Dawes, and T. R. Klaenhammer

The microbiologcal garden

www.mikrobiological-garden.net

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Lactobacteriaceae
• gram positive rods or cocci
• obligate fermenters (no respiratory chain)
• catalase negative (often aerotolerant)

Natural occurance
• Milk and milk products, fruit juice,
plant products, intestine, mucosa

Play an important role for

• production of curdled milk products

also: Sauerkraut and salami

www.microbiological-garden.net

Lactobacteriaceae classified by:

shape (cocci or rods) and type of fermentation

homolactic heterolactic
cocci rods cocci rods
Lactococcus Lactobacillus Leuconostoc Lactobacillus
L. lactis L. plantarum L. mesenteroides L. brevis
L. casei L. bulgaricus L. dextranicum L. kandleri
L. acidophilus
Enterococcus
E. faecalis
Streptococcus
S. thermophilus
S. salivarius
S. mutans
S. pyogenes

mainly lactate different fermentation products

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The general priciple of fermentation

glucose

ATP
reduction
NADH equivalents
respiratory chain
2 pyruvate
CO2, NADH
ATP organic substrate

degradation
ATP
CO2
intermediates
GTP
[H]
FADH2
NADH oxidised reduced
products products

Homolactic fermentation

COOH
C O
6 ATP
CH3
glucose 2 pyruvate

2 NAD+ 2 NADH

2 lactate
Lactate dehydrogenase
COOH
HC OH
CH3

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Heterolactic fermentation

Fig.: Schlegel. (1992)

Mixed acids fermentation

Products after fermentation of glucose (e.g. E. coli)
mol per100 mol Glucose

2,3-Butanediol CH3-CHOH-CHOH-CH3 0

Ethanol CH3-CH2 OH 42

Succinate COOH-CH2-CH2-COOH 29

Lactate CH3-CHOH-COOH 84

Acetate CH3-COOH 44

Formiate HCOOH 2

Hydrogen H2 43

Carbon dioxide CO2 44 after: Thimann (1955)

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Mixed acids fermentation

glykolysis
glucose pyruvate lactate

CO2

succinate
Ethanol CH3-CH2 OH

Succinate COOH-CH2-CH2-COOH
ethanol
Lactate CH3-CHOH-COOH acetyl~CoA
acetate
Acetate CH3-COOH +
CO2
Formiate HCOOH formiate
H2
Hydrogen H2

Carbon dioxide CO2

Fig.: Brock (mod.)

„The horror scheme“

Fig.: Brock

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Where can we find fermenters in nature?

the anaerobic food web

The anaerobic primary fermenters

food web polymers secundary fermenters, syntrophs
sulfate reducers
methanogens
monomes

fatty acids, succinate,

alckohols, lactate

formiate, H2, acetate

CO2, methanol

CH4, CO2 CO2

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Where can we find fermenters in nature?

alimentary systems

General structure of the vertebrate alimentary system

mouth stomach
oesophagus hindgut or colon rectum
duodenum

cecum, post gastric

fermentation chamber

rumen,
pre gastric
fermentation chamber
Herbivoric vertebrates
• fermentation chamber for plant material
Ruminants (cow, sheep, camel)
• fermentation chamber (rumen) in front of the
stomach
Other herbivors (e.g. rodents, horse)
• between duodenum and colon
Some omnivors (e.g. human)
• strongly reduced (appendix)

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 Can we live without microbes?

Experiments on animal without intestinal flora

• aseptic breeding, no developement of gut flora
• high dosage of antibiotics, destruction of gut flora

As a general rule
• signs of strong underfeeding, often lethal
• herbivors can´t live at all without their gut flora

Why?

Vitamine excretion
Thiamine, Riboflavine, Pyridoxine, Vit. B12 und K
essential amino acids, ...

Homo sapiens
continuous increase of pH

stomach normaly free of bacteria

pH 1,5

102-103 cells·ml-1 in initial part

duodenum primarily Lactobacillus sp. and
pH 2-5 Enterococcus sp.

1-3·1011 cells·ml-1
e.g. Bacteroides, Bifidobacterium,
colon Enterococcus, Bifidobacterium,
pH 7
Peptococcus, Enterobacteriaceae, ...

Human faeces
• up to 30-50% bacterial biomass

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The rumen ecosystem
Enlargement of the oesophagus
Fermentation chamber (large volume) cow app. 100-250 l
sheep app. 6 l
residence time 9-12 h

Physico-chemical conditions
pH 5,5 - 6,9 (mean: 6,4)
temperature 37-42°C
dry mass 10-18 %
redox potential -350 to -400 mV
gas phase 65 % CO2, 27 % CH4, 7 % N2, 0,6 % O2, 0,2 % H2
dissolved fatty acids 68 mM acetate, 20 mM propionate, 10 mM butyrate, 2 mM FA > C4
ammonium 2-12 mM

Biology
prokaryontes 1010 - 1011 g-1 (more than 200 species)
ciliates 104 - 106 g-1
fungy 102 - 104 g-1 (zoospores)

How does the cow eat?

Mouth: food is roughly hackled, swallowed, mixed with spittle
(bicarbonate buffered)

Rumen: mass is mixed thoroughly (muscle movement of rumen wall)

Reticulum: fibrous compounds are sieved, densified to chunks, refluxed and

ruminated

Omasum : water removal

duodenum rumen

Abdomasum: normal digestion

reticulum

oeso
phagus

abdomasum omasum
Fig.: Campbell und Reece 2003 (mod.)

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What happens in the rumen?
Fermentation of plant material
100 Glucose 113 acetate + 35 propionate + 26 butyrate + 104 CO2 + 61 CH4 + 43 H2O

starch cellulose pectine hemicelluloses

glucose fructose

pyruvate

CH4 acetate CO2 butyrate (lactate) propionate

What is the benefit for the cow?

• fermentation products (acetate, propionate and butyrate)

• bacterial biomass, gets into abdomasum after reflux
• N2 fixation in the rumen by anaerobic microorganisms

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What groups of microorganisms are found in the rumen?
Cellulose degrader Ruminococcus albus, Butyrivibrio fibrisolvens,
Fibrobacter succinogenes, Clostridium locheadii

Hemicellulose degrader Ruminococcus albus, Butyrivibrio fibrisolvens,

Fibrobacter succinogenes, Lachnospira multiparus
Sarch and sugar degrader Selenomonas ruminantium, Succinomonas amylolytica,
Bacteroides ruminicola, Streptococcus bovis

Lactate utiliser Selenomonas lactilytica, Megasphaera elsdenii,

Lac Prop + Ac Veillonella sp.

Succinate utiliser Selenomonas ruminantium, Veillonella parvula

Succ Prop + CO2

Methanogens Methanobrevibacter ruminantium,

CO2 + H2 CH4 Methanomicrobium mobile

Fungi and ciliates play a minor role: degradation of polymeric substances

Ciliates feed on bacteria: important for a stable microbial community

The termite gut

Wood feeding termites (e.g. Reticulitermes flavipes, app. 3 mm long)

have an enlarged hindgut as a fermentation chamber.

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Measurement of physico chemical parameter within the gut

embedding of gut in agarose (the tip of the microelectrode is marked)

Oxigen profiles
within the hindgut of
Reticulitermes flavipes

What happens in the termite gut?

polysaccharides from wood

protozoa

disolved disaccharides
and oligosaccharides acetate CO2, H2
fermenters
CH4
methanogens
CO2, H2, acetate, propionate, butyrate,
lactate, formiate homoacetogenic
bacteria

homoacetogenic
bacteria

absorption by termite

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