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

2. 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

3. 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

4. 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:
Smells like sauerkraut!

5. 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)

6. 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.

7. 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

8. 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.

9. 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

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

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

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

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

14. Thank you!