Fermentation. Glycolysis. ... when there is no external terminal electron acceptor! Substrate-level phosphorylation. Pyruvate. Fig.: Brock (mod.

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1 Fermentation... when there is no external terminal electron acceptor! Glycolysis Substrate-level phosphorylation 2 Pyruvate Fig.: Brock (mod.) 1

2 The TCA cycle glucose ATP NADH 2 pyruvate CO 2, NADH ATP reduction equivalents respiratory chain CO 2 GTP FADH 2 NADH The general priciple of fermentation The problem Regeneration of NADH 2 to NAD + glucose ATP reduction NADH equivalents 2 pyruvate CO 2, NADH ATP respiratory chain CO 2 GTP FADH 2 NADH The solution Transfer of reduction equivalents [H] on intermediates (e.g. pyruvate) or co-substrates 2

3 The general priciple of fermentation organic substrate ATP degradation 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 3

4 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 4

5 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 Lactobacteriaceae classified by: shape (cocci or rods) and type of fermentation homolactic cocci rods Lactococcus Lactobacillus L. lactis L. plantarum L. casei L. bulgaricus L. acidophilus Enterococcus E. faecalis Streptococcus S. thermophilus S. salivarius S. mutans S. pyogenes mainly lactate heterolactic cocci rods Leuconostoc Lactobacillus L. mesenteroides L. brevis L. dextranicum L. kandleri different fermentation products 5

6 The general priciple of fermentation glucose ATP NADH 2 pyruvate CO 2, NADH ATP reduction equivalents respiratory chain organic substrate degradation CO 2 ATP intermediates GTP [H] FADH 2 NADH oxidised products reduced products Homolactic fermentation glucose 2 ATP COOH C O CH 3 2 pyruvate 2 NAD + 2 NADH 2 lactate COOH HC OH Lactate dehydrogenase CH 3 6

7 Heterolactic fermentation NADH 2 NADH 2 NADH 2 NADH 2 Fig.: Schlegel. (1992) Heterolactic fermentation NADH 2 NADH 2 NADH 2 NADH 2 NADH 2 NADH 2 Fig.: Schlegel. (1992) 7

8 Mixed acids fermentation Products after fermentation of glucose (e.g. E. coli) mol per100 mol Glucose 2,3-Butanediol CH 3 -CHOH-CHOH-CH 3 0 Ethanol CH 3 -CH 2 OH 42 Succinate COOH-CH 2 -CH 2 -COOH 29 Lactate CH 3 -CHOH-COOH 84 Acetate CH 3 -COOH 44 Formiate HCOOH 2 Hydrogen H 2 43 Carbon dioxide CO 2 44 after: Thimann (1955) Mixed acids fermentation glucose glycolysis pyruvate lactate CO 2 Ethanol CH 3 -CH 2 OH succinate Succinate COOH-CH 2 -CH 2 -COOH Lactate CH 3 -CHOH-COOH Acetate CH 3 -COOH Formiate HCOOH Hydrogen H 2 acetyl~coa + formiate ethanol acetate CO 2 H 2 Carbon dioxide CO 2 Fig.: Brock (mod.) 8

9 Mixed acids fermentation and ATP = 2.5 ATP Where can we find fermenters in nature? the anaerobic food web 9

10 The anaerobic food web polymers monomes primary fermenters secundary fermenters, syntrophs sulfate reducers methanogens fatty acids, succinate, alckohols, lactate formiate, H 2, CO 2, methanol acetate CH 4, CO 2 CO 2 Where can we find fermenters in nature? alimentary systems 10

11 General structure of the vertebrate alimentary system mouth oesophagus stomach 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 Why? As a general rule signs of strong underfeeding, often lethal herbivors can t live at all without their gut flora Vitamine excretion thiamine, riboflavine, pyridoxine, vit. B 12 and K essential amino acids,... 11

12 Homo sapiens continuous increase of ph stomach ph 1,5 normaly free of bacteria duodenum ph cells ml -1 in initial part primarily Lactobacillus sp. and Enterococcus sp. colon ph 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 app l sheep app. 6 l Residence time 9-12 h Physico-chemical conditions ph 5,5-6,9 (mean: 6,4) temperature C dry mass % redox potential -350 to -400 mv gas phase 65 % CO 2, 27 % CH 4, 7 % N 2, 0,6 % O 2, 0,2 % H 2 dissolved fatty acids 68 mm acetate, 20 mm propionate, 10 mm butyrate, 2 mm FA > C 4 ammonium 2-12 mm Biology prokaryontes g -1 (more than 200 species) ciliates g -1 fungy g -1 (zoospores) 12

13 How does the cow eat? Mouth: Rumen: Reticulum: food is roughly hackled, swallowed, mixed with spittle (bicarbonate buffered) mass is mixed thoroughly (muscle movement of rumen wall) fibrous compounds are sieved, densified to chunks, refluxed and ruminated Omasum: water removal duodenum rumen Abdomasum: normal digestion reticulum oeso phagus abdomasum Fig.: Campbell und Reece 2003 (mod.) omasum What happens in the rumen? Fermentation of plant material 100 Glucose 113 acetate + 35 propionate + 26 butyrate CO CH H 2 O starch cellulose pectine hemicelluloses glucose fructose pyruvate CH 4 acetate CO 2 butyrate (lactate) propionate 13

14 What is the benefit for the cow? fermentation products (acetate, propionate and butyrate) bacterial biomass, gets into abdomasum after reflux N 2 fixation in the rumen by anaerobic microorganisms What groups of microorganisms are found in the rumen? Cellulose degrader Hemicellulose degrader Sarch and sugar degrader Ruminococcus albus, Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Clostridium locheadii Ruminococcus albus, Butyrivibrio fibrisolvens, Fibrobacter succinogenes, Lachnospira multiparus Selenomonas ruminantium, Succinomonas amylolytica, Bacteroides ruminicola, Streptococcus bovis Lactate utiliser Selenomonas lactilytica, Megasphaera elsdenii, Lac Prop + Ac Veillonella sp. Succinate utiliser Succ Prop + CO 2 Selenomonas ruminantium, Veillonella parvula Methanogens Methanobrevibacter ruminantium, CO 2 + H 2 CH 4 Methanomicrobium mobile Fungi and ciliates play a minor role: Ciliates feed on bacteria: degradation of polymeric substances important for a stable microbial community 14

15 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 15

16 What happens in the termite gut? polysaccharides from wood protozoa disolved disaccharides and oligosaccharides fermenters CO 2, H 2, acetate, propionate, butyrate, lactate, formiate acetate,co 2, H 2 homoacetogenic bacteria methanogens CH 4 homoacetogenic bacteria absorption by termite 16