1. Dusty Berggren 07.22.10 Dissimilatory Sulfate Reducers (SRBs)

2. Introduction Sulfate reducers… reduce sulfate to sulfide (8 e - transferred total) are strict anaerobes can act as syntrophs Use H 2, formate, acetate, propionate, lactate, ethanol, butyrate… as electron donors Oxidize organic compounds to CO 2 (complete) or a mixture of CO 2 and acetate (incomplete) can produce a hydrogen sulfide smell or FeS precipitates (black)  G o ’ = -44.5 kJ/mol If ATP  2P i Desulfovibrio sapovorans www.microbiological-garden.net

3. Enrichments ComponentPurpose --Basal Marine Mineral Media -- Na 2 SO 4 (30 mM)Electron Acceptor Sodium Lactate (10 mM)Electron Donor Resazurin (x 1000)Anaerobic Indicator NaHCO 3 (30 mM)pH Buffer 7 Vitamin-SolutionVitamins for growth SL 10Trace Element Solution Na 2 S (1 mM) Reductant to maintain anaerobic conditions pH  7.2 Inoculate 50 mL Pfennig bottles with sediment sample Store in dark at 30 o C Monitor development of sulfide smell (<3 days) Observe microscopically Transfer ~3 mL liquid to new media bottle when necessary Enrich by dilution

4. Enrichments ComponentPurpose --Basal Marine Mineral Media -- Na 2 SO 4 (30 mM)Electron Acceptor Sodium Lactate (10 mM)Electron Donor Resazurin (x 1000)Anaerobic Indicator NaHCO 3 (30 mM)pH Buffer 7 Vitamin-SolutionVitamins for growth SL 10Trace Element Solution Na 2 S (1 mM) Reductant to maintain anaerobic conditions pH  7.2 Selecting for organisms that… are anaerobes (facultative or obligate) can utilize SO 4 2- as an electron acceptor or ferment lactate (SRBs or syntrophs… may be one in the same) grow around neutral pH

5. The Poor-Man’s SRB Picture

6. The Rich-Man’s SRB Picture

7. Morphology General Consensus… Sulfate reducers aren’t very fun to take pictures of. --Small rods or ‘blobs’--

8. Further Enrichment  Agar Shakes

9. Deep Agar Dilution Series “Shakes” 3 mL 3% Agar 6 mL SRB Media ~1 mL of culture/media from enrichment bottle Anoxic headspace Single colonies can be extracted and introduced to a Pfennig bottle with fresh SRB media for further purification

10. The Point? Knowing an organisms function aids its enrichment and purification through manipulation of the physical/chemical environment