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Fermentation
... when there is no external terminal electron acceptor!
Substrate-level phosphorylation
Pyruvate2
Glycolysis
Fig.: Brock (mod.)
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glucose
2 pyruvate
GTP
NADH
ATP
NADH
FADH2
ATP
CO2, NADH
CO2
reduction
equivalentsrespiratory chain
The calvin cycle
glucose
2 pyruvate
GTP
NADH
ATP
NADH
FADH2
ATP
CO2, NADH
CO2
reduction
equivalentsrespiratory chain
The general priciple of fermentation
The problem Regeneration of
NADH2 to NAD+
The solutionTransfer of reduction equivalents [H] on intermediates
(e.g. pyruvate) or co-substrates
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Drawback Excretion of energy rich (reduced) substrates (e.g. ethanol)
reduced
products
organic substrate
[H]
ATP
degradation
intermediates
oxidisedproducts
The general priciple of fermentation
Bacterial fermentations are named by
their characteristical end productsalcohol (Ethanol) lactic acidbutyric acid propionic acidmixture of different acids
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
The general priciple of fermentation
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e.g.Lactobacillus spec.
Lactobacteriaceaehomolactic fermentation
Photo: M. Dykstra, R. Barrangou,R. Sanozky-Dawes, and T. R. Klaenhammer
The easiest fermentative pathway
... a bit more complicated:
heterolactic fermentation
The microbiologcal garden
www.mikrobiological-garden.net
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Natural occurance
Milk and milk products, fruit juice,
plant products, intestine, mucosa
Lactobacteriaceae
gram positive rods or cocci obligate fermenters (no respiratory chain)
catalase negative (often aerotolerant)
www.microbiological-garden.net
Play an important role for
production of curdled milk products
also: Sauerkraut and salami
Lactobacteriaceae classified by:
shape (cocci or rods) and type of fermentation
homolactic
cocci rods
Lactococcus Lactobacillus
L. lactis L. plantarumL. casei L. bulgaricus
L. acidophilusEnterococcus
E. faecalis
StreptococcusS. thermophilus
S. salivarius
S. mutans
S. pyogenes
mainly lactate
heterolactic
cocci rods
Leuconostoc Lactobacillus
L. mesenteroides L. brevisL. dextranicum L. kandleri
different fermentation products
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glucose
2 pyruvate
GTP
NADH
ATP
NADH
FADH2
ATP
CO2, NADH
CO2
reduction
equivalentsrespiratory chain
reducedproducts
organic substrate
[H]
ATP
degradation
intermediates
oxidised
products
The general priciple of fermentation
glucose 2 pyruvate
2 NADH2 NAD+
ATP6
COOH
C O
CH3
Lactate dehydrogenase2 lactate
COOH
HC OH
CH3
Homolactic fermentation
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Fig.: Schlegel. (1992)
Heterolactic fermentation
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)
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Ethanol CH3-CH2OH
Succinate COOH-CH2-CH2-COOH
Lactate CH3-CHOH-COOH
Acetate CH3-COOH
Formiate HCOOH
Hydrogen H2
Carbon dioxide CO2
glucoseglykolysis
pyruvate lactate
acetyl~CoA
formiate
+
ethanol
acetate
CO2
H2
CO2
succinate
Mixed acids fermentation
Fig.: Brock (mod.)
The horror scheme
Fig.: Brock
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Where can we find fermenters in nature?
the anaerobic food web
The anaerobicfood web
CH4, CO2CO2
secundary fermenters, syntrophs
methanogens
sulfate reducers
primary fermenters
formiate, H2,CO2, methanol
fatty acids, succinate,
alckohols, lactate
acetate
polymers
monomes
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Where can we find fermenters in nature?
alimentary systems
mouth stomachhindgut or colon rectumoesophagus duodenum
rumen,pre gastricfermentation chamber
cecum, post gastricfermentation 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)
General structure of the vertebrate alimentary system
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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
Why?
As a general rule signs of strong underfeeding, often lethal herbivors cant live at all without their gut flora
Vitamine excretionThiamine, Riboflavine, Pyridoxine, Vit. B12 und Kessential amino acids, ...
Homo sapiens
stomachpH 1,5
duodenumpH 2-5
colonpH 7
normaly free of bacteria
102-103 cellsml-1 in initial partprimarily Lactobacillussp. andEnterococcussp.
1-31011 cellsml-1
e.g. Bacteroides, Bifidobacterium,Enterococcus, Bifidobacterium,Peptococcus, Enterobacteriaceae, ...
Human faeces up to 30-50% bacterial biomass
continuous increase of pH
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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 conditionspH 5,5 - 6,9 (mean: 6,4)temperature 37-42Cdry mass 10-18 %redox potential -350 to -400 mVgas phase 65 % CO2, 27 % CH4, 7 % N2, 0,6 % O2, 0,2 % H2dissolved fatty acids 68 mM acetate, 20 mM propionate, 10 mM butyrate, 2 mM FA > C 4ammonium 2-12 mM
Biologyprokaryontes 1010 - 1011 g-1 (more than 200 species)ciliates 104 - 106 g-1
fungy 102 - 104 g-1 (zoospores)
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 andruminated
Omasum : water removal
Abdomasum: normal digestion
How does the cow eat?
duodenum
reticulum
oesophagus
omasumabdomasum
Fig.: Campbell und Reece 2003 (mod.)
rumen
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starch cellulose pectine hemicelluloses
glucose fructose
pyruvate
CH4 acetate CO2 butyrate (lactate) propionate
What happens in the rumen?
Fermentation of plant material100 Glucose 113 acetate + 35 propionate + 26 butyrate+ 104 CO2 + 61 CH4 + 43 H2O
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 parvulaSucc Prop + CO2
Methanogens Methanobrevibacter ruminantium,CO2 + H2 CH4 Methanomicrobium mobile
Fungi and ciliates play a minor role: degradation of polymeric substancesCiliates feed on bacteria: important for a stable microbial community
Wood feeding termites (e.g. Reticulitermes flavipes, app. 3 mm long)
have an enlarged hindgut as a fermentation chamber.
The termite gut
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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
polysaccharides from wood
disolved disaccharidesand oligosaccharides
homoacetogenicbacteria
CO2, H2, acetate, propionate, butyrate,
lactate, formiate
fermenters
protozoa
absorption by termite
CH4
homoacetogenicbacteria
methanogens
CO2, H2acetate
What happens in the termite gut?