Fermentation
Glycolysis
Substrate-level phosphorylation
2
Pyruvate
Fig.: Brock (mod.)
The TCA cycle
glucose
ATP
NADH
reduction
equivalents
respiratory chain
2 pyruvate
CO2, NADH
ATP
CO2
GTP
FADH2
NADH
The general priciple of fermentation
The problem
• Regeneration of
NADH2 to NAD+
glucose
ATP
NADH
reduction
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 http://www.youtube.com/watch?v=StXlo1W3Gvg
The general priciple of fermentation
organic substrate
degradation
ATP
intermediates
[H]
oxidised
products
reduced
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
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
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
L. lactis
L. casei
Lactobacillus
L. plantarum
L. bulgaricus
L. acidophilus
Leuconostoc
Lactobacillus
L. mesenteroides L. brevis
L. dextranicum L. kandleri
Enterococcus
E. faecalis
Streptococcus
S. thermophilus
S. salivarius
S. mutans
S. pyogenes
mainly lactate
different fermentation products
The general priciple of fermentation
glucose
ATP
NADH
reduction
equivalents
respiratory chain
2 pyruvate
CO2, NADH
ATP
organic substrate
degradation
ATP
CO2
intermediates
GTP
[H]
FADH2
NADH
oxidised
products
Homolactic fermentation
COOH
C
6 ATP
CH3
glucose
2 pyruvate
2 NAD+
2 lactate
COOH
HC OH
CH3
O
2 NADH
Lactate dehydrogenase
reduced
products
Heterolactic fermentation
NADH2
NADH2
NADH2
NADH2
NADH2
NADH2
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-CH2OH
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)
Mixed acids fermentation
glycolysis
glucose
pyruvate
lactate
CO2
Ethanol
CH3-CH2OH
Succinate
COOH-CH2-CH2-COOH
Lactate
CH3-CHOH-COOH
Acetate
CH3-COOH
Formiate
HCOOH
Hydrogen
H2
succinate
ethanol
acetyl~CoA
acetate
+
CO2
formiate
H2
Carbon dioxide CO2
Fig.: Brock (mod.)
Where can we find fermenters in nature?
the anaerobic food web
The anaerobic
food web
primary fermenters
polymers
secundary fermenters, syntrophs
sulfate reducers
monomes
methanogens
fatty acids, succinate,
alckohols, lactate
formiate, H2,
CO2, methanol
acetate
CH4, CO2
CO2
An example from the Wadden sea
Combination of Stable-Isotope Probing and
Microcalorimetry to identify fermenting bacteria
Key questions
1. Which microorganisms are involved in the different steps
of the degradation process?
2. What are the predominant fermentation pathways?
3.Which are the intermediate substrates?
Æ Accumulation by inhibition experiments
Microcalorimetry
Heat production as a criterion for
metabolic activity
Stable Isotope Probing
Who does what?
1.13C-organic matter incorporation
13C
☺
13C
13C
2. Extraction of DNA/RNA and
ribosomes?
3. Density-gradient centrifugation
4. Characterisation by gene
probing and sequence analysis
SIP links function to
identification without
cultivation !!!
12C-RNA
13C-RNA
First experiment: degradation of 13C-glucose
Sampling after 22h
Heat Production [mW]
Sampling after 75h
Time [h]
Detection of fermentation pathways
Untreated at Tp.
0
1.Tp.
2.Tp.
Glucose [mM]
not detectable
4.70
not detectable
Lactate [mM]
not detectable
0.35
not detectable
Formate [mM]
0.20
8.67
2.02
Acetate [mM]
0.27
16.20
38.90
Propionate
[mM]
not detectable
0.33
2.81
Sulfate [mM]
4.49E-02
9.90E-04
9.90E-04
→pure culture: to identify the most important degradation pathways
Distribution of 12C- and 13C-glucose in the density gradient
Ratio of maximum quantities
12C
13C
1.0
0.8
0.6
0.4
0.2
0
1.76
1.78
1.80
1.82
CsTFA-buoyant density [g
c
1.84
ml-1]
1.86
1. tp, 12C-glucose
2. tp, 12C-glucose
1. tp, 13C-glucose
2. tp, 13C-glucose
A Stenotrophomonas maltophilia related bacterium
is the main degrader of glucose
Buoyant density
M high
low
Closest cultiv. relative
Sim. [%]
Affiliation
Beta proteobacterium
Burkholderia sp.
99
99
β-Proteobacteria
β-Proteobacteria
Delftia acidovorans
97
Burkholderia sp.
90
Stenotrophomonas maltophilia 93
β-Proteobacteria
β-Proteobacteria
γ-Proteobacteria
Rhizosphere soil bact.
Stenotrophomonas sp.
Leptothrix ginsengisoli
89
97
90
γ-Proteobacteria
γ-Proteobacteria
β-Proteobacteria
Rhizosphere soil bact.
Rhodovulum marinum
95
95
γ-Proteobacteria
α-Proteobacteria
Phycicoccus jejuensis
Arthrobacter sp.
Phycicoccus dokdonensis
97
98
97
Actinobacteria
Actinobacteria
Actinobacteria
Where else can we find
fermenters in nature?
alimentary systems
General structure of the vertebrate alimentary system
mouth
stomach
oesophagus
duodenum
hindgut or colon
rectum
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)
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 and K
essential amino acids, ...
Homo sapiens
continuous increase of pH
stomach
pH 1,5
duodenum
pH 2-5
colon
pH 7
normaly free of bacteria
102-103 cells·ml-1 in initial part
primarily Lactobacillus sp. and
Enterococcus sp.
1-3·1011 cells·ml-1
e.g. Bacteroides, Bifidobacterium,
Enterococcus, Bifidobacterium,
Peptococcus, Enterobacteriaceae, ...
Human faeces
• up to 30-50% bacterial biomass
The rumen ecosystem
Enlargement of the oesophagus
Fermentation chamber (large volume)
cow
sheep
Residence time
9-12 h
app. 100-250 l
app. 6 l
Physico-chemical conditions
pH
temperature
dry mass
redox potential
gas phase
dissolved fatty acids
ammonium
5,5 - 6,9 (mean: 6,4)
37-42°C
10-18 %
-350 to -400 mV
65 % CO2, 27 % CH4, 7 % N2, 0,6 % O2, 0,2 % H2
68 mM acetate, 20 mM propionate, 10 mM butyrate, 2 mM FA > C4
2-12 mM
Biology
prokaryontes
ciliates
fungy
1010 - 1011 g-1 (more than 200 species)
104 - 106 g-1
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
omasum
abdomasum
Fig.: Campbell und Reece 2003 (mod.)
What happens in the rumen?
Fermentation of plant material
100 Glucose
113 acetate + 35 propionate + 26 butyrate + 104 CO2 + 61 CH4 + 43 H2O
starch
pectine
cellulose
glucose
hemicelluloses
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
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,
Veillonella sp.
Lac
Prop + Ac
Succinate utiliser
Succ
Selenomonas ruminantium, Veillonella parvula
Prop + CO2
Methanogens
CO2 + H2
CH4
Methanobrevibacter ruminantium,
Methanomicrobium mobile
Fungi and ciliates play a minor role:
Ciliates feed on bacteria:
degradation of polymeric substances
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.
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
methanogens
CO2, H2, acetate, propionate, butyrate,
lactate, formiate
CH4
homoacetogenic
bacteria
homoacetogenic
bacteria
absorption by termite
Fermentation
... when there is no external terminal electron acceptor!