Fermentation

Substrate-level phosphorylation

Pyruvate2

Glycolysis

Fig.: Brock (mod.)

glucose

2 pyruvate

GTP

NADH

ATP

NADH

FADH2

ATPCO2, NADH

CO2

reductionequivalents

respiratory chain

The TCA cycle

glucose

2 pyruvate

GTP

NADH

ATP

NADH

FADH2

ATPCO2, NADH

CO2

reductionequivalents

respiratory chain

The general priciple of fermentation

The solution•Transfer of reduction equivalents [H] on intermediates

(e.g. pyruvate) or co-substrates

The problem• Regeneration of

NADH2 to NAD+

http://www.youtube.com/watch?v=StXlo1W3Gvg

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

reducedproducts

organic substrate

[H]

ATPdegradation

intermediates

oxidised products

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

homolactic fermentation

e.g.Lactobacillus spec.

Lactobacteriaceae

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

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

homolacticcocci rods

Lactococcus LactobacillusL. lactis L. plantarumL. casei L. bulgaricus

L. acidophilusEnterococcusE. faecalis

StreptococcusS. thermophilusS. salivariusS. mutansS. pyogenes

mainly lactate

heterolacticcocci rods

Leuconostoc LactobacillusL. mesenteroides L. brevisL. dextranicum L. kandleri

different fermentation products

glucose

2 pyruvate

GTP

NADH

ATP

NADH

FADH2

ATPCO2, NADH

CO2

reductionequivalents

respiratory chain

reducedproducts

organic substrate

[H]

ATPdegradation

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

Heterolactic fermentation

Fig.: Schlegel. (1992)

NADH2 NADH2

NADH2NADH2

NADH2

NADH2

Mixed acids fermentationProducts 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 44after: Thimann (1955)

glucoseglycolysis

pyruvate lactate

acetyl~CoA

formiate

+

ethanol

acetate

CO2

H2

CO2

succinateEthanol CH3-CH2OH

Succinate COOH-CH2-CH2-COOH

Lactate CH3-CHOH-COOH

Acetate CH3-COOH

Formiate HCOOH

Hydrogen H2

Carbon dioxide CO2

Mixed acids fermentation

Fig.: Brock (mod.)

Where can we find fermenters in nature?

the anaerobic food web

The anaerobic food web

CH4, CO2CO2

secundary fermenters, syntrophs

methanogens

sulfate reducers

primary fermenters

formiate, H2,CO2, methanol

fatty acids, succinate,alckohols, lactate

acetate

polymers

monomes

Combination of Stable-Isotope Probing and Microcalorimetry to identify fermenting bacteria

An example from the Wadden sea

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?

Accumulation by inhibition experiments

Key questions

Microcalorimetry

Heat production as a criterion formetabolic activity

Stable Isotope Probing

1.13C-organic matter

Who does what?

incorporation

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-RNA13C-RNA

13C

13C13C☺

Hea

t Pro

duct

ion

[mW

]

Time [h]

First experiment: degradation of 13C-glucose

Sampling after 22h

Sampling after 75h

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

c

CsTFA-buoyant density [g ml-1]

Rat

io o

f max

imum

qua

ntiti

es

0

0.2

0.4

0.6

0.8

1.0

1.76 1.78 1.80 1.82 1.84

12C 13C

1. tp, 12C-glucose2. tp, 12C-glucose1. tp, 13C-glucose2. tp, 13C-glucose

1.86c

CsTFA-buoyant density [g ml-1]

Rat

io o

f max

imum

qua

ntiti

es

0

0.2

0.4

0.6

0.8

1.0

1.76 1.78 1.80 1.82 1.84

12C 13C

1. tp, 12C-glucose2. tp, 12C-glucose1. tp, 13C-glucose2. tp, 13C-glucose

1. tp, 12C-glucose2. tp, 12C-glucose1. tp, 13C-glucose2. tp, 13C-glucose

1.86

Distribution of 12C- and 13C-glucose in the density gradient

A Stenotrophomonas maltophilia related bacteriumis the main degrader of glucose

Sim. [%]Closest cultiv. relative Affiliation

Burkholderia sp. β-Proteobacteria99

Stenotrophomonas maltophilia γ-Proteobacteria93

Stenotrophomonas sp. γ-Proteobacteria97

Rhodovulum marinum α-Proteobacteria95

Arthrobacter sp. Actinobacteria98

Delftia acidovorans β-Proteobacteria97Burkholderia sp. β-Proteobacteria90

Leptothrix ginsengisoli β-Proteobacteria90

Phycicoccus jejuensis Actinobacteria97

Phycicoccus dokdonensis Actinobacteria97

Beta proteobacterium β-Proteobacteria99

Rhizosphere soil bact. 89 γ-Proteobacteria

95Rhizosphere soil bact. γ-Proteobacteria

M high lowBuoyant density

Sim. [%]Closest cultiv. relative Affiliation

Burkholderia sp. β-Proteobacteria99

Stenotrophomonas maltophilia γ-Proteobacteria93

Stenotrophomonas sp. γ-Proteobacteria97

Rhodovulum marinum α-Proteobacteria95

Arthrobacter sp. Actinobacteria98

Delftia acidovorans β-Proteobacteria97Burkholderia sp. β-Proteobacteria90

Leptothrix ginsengisoli β-Proteobacteria90

Phycicoccus jejuensis Actinobacteria97

Phycicoccus dokdonensis Actinobacteria97

Beta proteobacterium β-Proteobacteria99

Rhizosphere soil bact. 89 γ-Proteobacteria

95Rhizosphere soil bact. γ-Proteobacteria

M high lowBuoyant density

Where else can we findfermenters in nature?

alimentary systems

mouth stomach hindgut or colon rectumoesophagus duodenum

rumen,pre gastric fermentation chamber

cecum, post gastric fermentation chamber

Herbivoric vertebrates• fermentation chamber for plant material

Ruminants (cow, sheep, camel)• fermentation chamber (rumen) in front of the

stomach

General structure of the vertebrate alimentary system

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

Why?

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

Vitamine excretionthiamine, riboflavine, pyridoxine, vit. B12 and Kessential amino acids, ...

Homo sapiens

normaly free of bacteria

102-103 cells·ml-1 in initial partprimarily Lactobacillus sp. andEnterococcus sp.

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

Human faeces• up to 30-50% bacterial biomass

stomachpH 1,5

duodenumpH 2-5

colonpH 7

continuous increase of pH

The rumen ecosystemEnlargement of the oesophagus

Fermentation chamber (large volume) cow app. 100-250 lsheep app. 6 l

Residence time 9-12 h

Physico-chemical conditionspH 5,5 - 6,9 (mean: 6,4)temperature 37-42°Cdry 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 > C4ammonium 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 and ruminated

Omasum: water removal

Abdomasum: normal digestion

How does the cow eat?

duodenum

reticulum

oesophagus

omasumabdomasumFig.: Campbell und Reece 2003 (mod.)

rumen

starch cellulose pectine hemicelluloses

glucose fructose

pyruvate

CH4 acetate CO2 butyrate (lactate) propionate

What happens in the rumen?Fermentation of plant material

100 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

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

Measurement of physico chemical parameter within the gut

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

Oxigen profileswithin the hindgut of Reticulitermes flavipes

polysaccharides from wood

disolved disaccharides and 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?

Fermentation

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