Dusty Berggren07.22.10

Dissimilatory Sulfate Reducers(SRBs)

IntroductionSulfate reducers…

reduce sulfate to sulfide (8 e- transferred total)are strict anaerobescan act as syntrophs

Use H2, formate, acetate, propionate, lactate, ethanol, butyrate… as electron donors

Oxidize organic compounds to CO2 (complete) or a mixture of CO2 and acetate (incomplete)

can produce a hydrogen sulfide smell or FeS precipitates (black)

23

][224 )( SOAPSsulfateinephosphoAdenosSO

reductaseAPS

H

esulfurylasATP

ATP

HSHSOreductaseBisulfite

H ][63

GGoo’ = -44.5 ’ = -44.5 kJ/molkJ/mol

If ATP If ATP 2P 2Pii

Desulfovibrio sapovorans

www.microbiological-garden.net

EnrichmentsComponent Purpose

--Basal Marine Mineral Media --

Na2SO4 (30 mM) Electron Acceptor

Sodium Lactate (10 mM)

Electron Donor

Resazurin (x 1000)

Anaerobic Indicator

NaHCO3 (30 mM) pH Buffer

7 Vitamin-Solution

Vitamins for growth

SL 10Trace Element Solution

Na2S (1 mM)

Reductant to maintain anaerobic conditions

pH 7.2

Inoculate 50 mL Pfennig bottles with sediment sample

Store in dark at 30oCMonitor development

of sulfide smell (<3 days)

Observe microscopically

Transfer ~3 mL liquid to new media bottle when necessaryEnrich by dilution

EnrichmentsComponent Purpose

--Basal Marine Mineral Media --

Na2SO4 (30 mM) Electron Acceptor

Sodium Lactate (10 mM)

Electron Donor

Resazurin (x 1000)

Anaerobic Indicator

NaHCO3 (30 mM) pH Buffer

7 Vitamin-Solution

Vitamins for growth

SL 10Trace Element Solution

Na2S (1 mM)

Reductant to maintain anaerobic conditions

pH 7.2

Selecting for organisms that…

are anaerobes (facultative or

obligate)

can utilize SO42- as an

electron acceptor or ferment

lactate(SRBs or syntrophs…

may be one in the same)

grow around neutral pH

The Poor-Man’s SRB Picture

The Rich-Man’s SRB Picture

MorphologyGeneral

Consensus…

Sulfate reducers aren’t very fun to take pictures of.

--Small rods or ‘blobs’--

Further Enrichment Agar Shakes

Deep Agar Dilution Series “Shakes”

3 mL 3% Agar6 mL SRB Media

~1 mL of culture/media from enrichment bottleAnoxic headspace

Single colonies can be extracted and introduced to a

Pfennig bottle with fresh SRB media for further purification

The Point?

Knowing an organisms function aids its enrichment and purification

through manipulation of the physical/chemical environment