Lactic Acid Bacteria and Propionic Acid Bacteria Enrichments Julie Huang Hopkins Microbiology Course July 15, 2011

Lactic Acid Bacteria General Features: Metabolism: Gram +, acid-tolerant, halotolerant, aerotolerant, rod or cocci Metabolism: Glucose  2 pyruvate  lactate Homofermentative Glucose → 2 lactate Heterofermentative Glucose → lactate + ethanol + carbon dioxide Rods and cocci-lactobacillus and leuconostoc (ovid cocci often forming chains). Picture is of sauerkraut juice inoculum therefore a mix of lactic acid bacteria Metabolism- glucose converted to pyruvate via glycolysis which yields 2 ATP and 2 reducing equivalents (NADH). Pyruvate is then reduced with the reducing equivalents to form lactic acid, ethanol, and carbon dioxide is evolved. Heterofermentative- lactic acid, ethanol, carbon dioxide Homofermentative- mostly lactic acid

Conditions for Enrichment Enrichment from sauerkraut High salinity- added 3% (w/w) NaCl to cabbage Anaerobic- added H20 and compressed cabbage layers Incubate jars at 30°C Isolation on agar plate Glucose and CaCO3 plates Dissolution of CaCO3 Glucose- carbon source for fermentation CaCO3 will dissolve with addition of acid and evolve carbon dioxide and water (fizz test for carbonates in geology) Glucose-CaCO3 plates Tryptone Glucose Yeast extract K2HPO4 CaCO3 agar

After 4 Days of Fermentation Streaked for isolation On glucose- CaCO3 plate Glucose-CaCO3 plates Tryptone Glucose Yeast extract K2HPO4 CaCO3 agar starting pH: 6.5-7 ending pH: 3.5-5.5 Smells like sauerkraut!

Lactic Acid Bacteria After Fermentation Leuconostoc is heterofermentative and often enriched in sauerkraut Kingdom: Bacteria Division: Firmicutes Class: Bacilli Order: Lactobacillales Family: Leuconostocaceae Genus: Leuconostoc (gram +, heterofermentative, resistant to vancomycin, catalase -) LAB from final isolation (100x) ID: Leuconostoc? (ovoid cocci and often form chains)

Propionic Acid Bacteria General Features: Gram +, non-motile, non-sporeforming, anaerobic, rod Metabolism: Lactate  propionate + acetate + carbon dioxide Brown colonies- requires B12 as part of their metabolism White colonies- use the acrylate pathway (no B12) Homofermentative lactic acid bacteria ferment lactose/glucose to lactate. Propionic acid bacteria then ferment lactate to propionate, acetate, CO2.

Conditions for Enrichment Enrichment from Swiss Cheese Fill screw capped tube with swiss cheese Add lactate yeast extract to cheese Bubble CO2 to bottom of tube for 30 seconds Incubate at 30°C in the dark Lactate Yeast Extract: 40g sodium lactate 5g yeast extract

Conditions for Enrichment Enrichment from Microbial Mat Why? Found evidence of lactate dehydrogenase in transcriptome study Found propionate, acetate, and formate, but not lactate in the mat Cyanobacteria ferment at night when competition for oxygen is high Inoculum Area underneath the cyanobacteria layer Inoculum Area underneath the cyanobacteria layer because the cyanobacteria may be producing lactate which creates a niche for proprionic acid bacteria.

After 4 Days of Fermentation (cheese inoculum) Brown and white colonies isolated from cheese Lactate- CaCO3 Agar Plates- Sodium-lactate Tryptone Yeast extract CaCO3 agar Lactate- CaCO3 plate

Propionic Acid Bacteria after Fermentation (cheese) Mostly rods (100x)

Enrichment Using Mat Inoculum Streak for isolation Lactate- CaCO3 Agar Plates- Sodium-lactate Tryptone Yeast extract CaCO3 agar

Propionic Acid Bacteria after Fermentation (mat) Silke’s enrichment White colonies- ovoid cocci (100x) Brown colonies-mostly rods (100x)

Catalase Test for LAB (-) and PAB (+) 2 H2O2 → 2 H2O + O2 Catalase can degrade 40 million hydrogen peroxide molecules/second Before peroxide After peroxide

Thank you!