Approaches and Techniques for Isolating and Cultivating Acidophiles
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Transcript of Approaches and Techniques for Isolating and Cultivating Acidophiles
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Approaches and Techniques for Isolating and Cultivating Acidophiles
D. Barrie Johnson
School of Biological Sciences,
University of Wales, Bangor,
LL57 2UW, U.K.
Bangor Acidophile Research Team
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What methods can (and should) be used to study “mine microbiology”?
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What methods can (and should) be used to study “mine microbiology”?
Culture-dependent methods
• enumeration• plate isolation• enrichment cultures• micromanipulation
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What methods can (and should) be used to study “mine microbiology”?
Culture-dependent methods
• enumeration• plate isolation• enrichment cultures• micromanipulation
Culture-independent methods
• PCR-dependent approaches
- clone libraries
- T-RFLP, DGGE etc.• PCR-independent
approaches
- FISH
- flow cytometry
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PCR 16S rRNA genes
T-RFLP analysis
New peak(s) observed
Clone library constructed & sequenced
Probe design: FISH analysis
Identification of unknown prokaryotes
Modification/redesign of media for isolating “unculturables ”
Isolation on solid media
Identifi cation of isolates from physiological traits and/or sequence analysis of 16S rRN A genes
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PCR 16S rRNA genes
T-RFLP analysis
New peak(s) observed
Clone library constructed & sequenced
Probe design: FISH analysis
Identification of unknown prokaryotes
Modification/redesign of media for isolating “unculturables ”
Isolation on solid media
Identification of isolates from physiological traits and/or sequence analysis of 16S rRN A genes
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PCR 16S rRNA genes
T-RFLP analysis
New peak(s) observed
Clone library constructed & sequenced
Probe design: FISH analysis
Identification of unknown prokaryotes
Modification/redesign of media for isolating “unculturables ”
Isolation on solid media
Identifi cation of isolates from physiological traits and/or sequence analysis of 16S rRN A genes
Identification of isolates from physiological traits and/or sequence analysis of 16S rRN A genes
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PCR 16S rRNA genes
T-RFLP analysis
New peak(s) observed
Clone library constructed & sequenced
Probe design: FISH analysis
Identification of unknown prokaryotes
Modification/redesign of media for isolating “unculturables ”
Isolation on solid media
Identifi cation of isolates from physiological traits and/or sequence analysis of 16S rRN A genes
Identification of isolates from physiological traits and/or sequence analysis of 16S rRNA genes
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PCR 16S rRNA genes
T-RFLP analysis
New peak(s) observed
Clone library constructed & sequenced
Probe design :
FISH analysis Identification of unknown prokaryotes
Modification/redesign of media for isolating “unculturables ”
Isolation on solid media
Identifi cation of isolates from physiological traits and/or sequence analysis of 16S rRN A genes
Quantitative
data
Identification of isolates from physiological traits and/or sequence analysis of 16S rRN A genes
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PCR 16S rRNA genes
T-RFLP analysis
New peak(s) observed
Clone library constructed & sequenced
Probe design: FISH analysis
Identification of unknown prokaryotes
Modification/redesign of media for isolating “unculturables ”
Isolation on solid media
Identifi cation of isolates from physiological traits and/or sequence analysis of 16S rRN A genes
Identification of isolates from physiological traits and/or sequence analysis of 16S rRN A genes
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Enumeration of microorganisms:
• Direct counts (phase contast microscopy; Thoma cell)
• Direct counts (stained cells)
• Most probable number (MPN) counts
• Plate counts
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Enumeration of microorganisms:
• Direct counts (phase contast microscopy; Thoma cell)
Advantages:
- minimum equipment requirement
- quick and easy
Disadvantages:
- minimum bacterial numbers ~106/ml
- prone to operator error
- not possible to differentiate/identify bacteria
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Enumeration of microorganisms:
• Direct counts (phase contast microscopy; Thoma cell)
• Direct counts (stained cells)
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Enumeration of microorganisms:
• Direct counts (stained cells) Advantages:
- accuracy
- possible to count low numbers of cells (adsorption onto membranes)
- can use e.g. DNA-specific dyes
Disadvantage
- not possible to differentiate/identify bacteria
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Trefriw biofilm stained with DAPI
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Enumeration of microorganisms:
• Direct counts (phase contast microscopy; Thoma cell)
• Direct counts (stained cells)
• Most probable number (MPN) counts
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Enumeration of microorganisms:
• Direct counts (phase contast microscopy; Thoma cell)
• Direct counts (stained cells)
• Most probable number (MPN) counts
• Plate counts
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Enumeration of microorganisms:
• Plate counts Advantages:
- extreme sensitivity (can count <10 bacteria/ml)
- can differentiate and aid preliminary identification of isolates
Disadvantage
- not all indigenous microorganisms may grow on solid media
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Problems with growing acidophiles on solid media
• Sensitivity of many acidophiles to organic materials in general and some materials (e.g. organic acids) in particular
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Problems with growing acidophiles on solid media
• Sensitivity of many acidophiles to organic materials in general and some materials (e.g. organic acids) in particular
• Purity of the gelling agent (e.g. agar)
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Problems with growing acidophiles on solid media
• Sensitivity of many acidophiles to organic materials in general and some materials (e.g. organic acids) in particular
• Purity of the gelling agent (e.g. agar)
wash agar before sterilization
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Problems with growing acidophiles on solid media
• Sensitivity of many acidophiles to organic materials in general and some materials (e.g. organic acids) in particular
• Purity of the gelling agent (e.g. agar)
• Hydrolysis of the gelling agent
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Problems with growing acidophiles on solid media
• Hydrolysis of the gelling agent
need for continuous removal of small molecular weight hydrolysates
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Early plate formulation: “FeTSB” medium
• Contains both ferrous iron and tryptone soya broth
• Designed to promote the growth of iron-oxidizing and heterotrophic acidophiles
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Acidophilic colonies: FeTSB medium
At. ferrooxidans
Acidiphilium sp.
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Dilution
Colonies nos. 10-3 10-4 10-5
Iron-oxidizers >103 200 0
Heterotrophs 80 8 0
FeTSB medium: typical data where numbers of iron-oxidizers > acidophilic heterotrophs
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Overlay plate technique for
isolating and enumerating
acidophilic microorganisms
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Overlay medium variants(Acidiphilium SJH in underlayer)
Code Energy sources pH Target isolates
Feo ferrous iron/(TSB) ~2.6 iron-oxidizers (heterotrophs)
FeSo ferrous iron, (TSB) ~2.6 iron-oxidizers tetrathionate sulfur-oxidizers (heterotrophs)
FeTo ferrous iron, (TSB) ~4.0 moderately acido- thiosulfate philic Fe & S- oxidizers and heterotrophs
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Acidophilic colonies: FeSo medium
At. thiooxidans
Ferrimicrobium
At. ferrooxidans
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Colonies of moderate acidophiles: FeTo medium
S-oxidizer
Thiomonas sp
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Isolation/enumeration of acidophilic heterotrophs
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Isolation/enumeration of acidophilic heterotrophs
• Extremely acidic environments are mostly oligotrophic (contain little organic C)
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Isolation/enumeration of acidophilic heterotrophs
• Extremely acidic environments are mostly oligotrophic (contain little organic C)
• acidophilic heterotrophs (like autotrophs) may be inhibited by medium-high concentrations of dissolved carbon, and very small amounts of organic acids
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Isolation/enumeration of acidophilic heterotrophs
• Extremely acidic environments are mostly oligotrophic (contain little organic C)
• acidophilic heterotrophs (like autotrophs) may be inhibited by medium-high concentrations of dissolved carbon, and very small amounts of organic acids
• overlay media again produce higher counts than non-overlay media
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Underlay heterotroph: Acidocella WJB3
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Underlay heterotroph: Acidocella WJB3
• Restricted metabolic capabilities
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Underlay heterotroph: Acidocella WJB3
• Restricted metabolic capabilities
• catabolizes organic acids (primary inhibitory compounds in solid media)
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Underlay heterotroph: Acidocella WJB3
• Restricted metabolic capabilities
• catabolizes organic acids (primary inhibitory compounds in solid media)
• does not grow on yeast extract or glycerol
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Overlay medium variants(Acidocella WJB3 in underlayer)
Code Energy sources pH Target isolates
YE3o yeast extract ~3.0 heterotrophs (extreme acidophiles) YE4o yeast extract ~4.0 heterotrophs (moderate acidophiles)
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Colonies of heterotrophic acidophiles: YE3o medium
Thiomonas s
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Case study 1: Roeros copper mine, Norway
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Roeros copper mine, Norway
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Acidophilic iron-oxidizers: Roeros copper mine, Norway
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Acidophilic heterotrophs: Roeros copper mine, Norway
Acidocella sp.
A.rubrum
Acidiphilium sp.
Acidobacterium sp.
Fratauria sp.
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SEXTUS MINE KING'S MINE
Outlet AMD Dump AMD Outlet AMD Fe-oxidizing bacteria
(total) 1.4 x 103 6.7 x 103 5.6 x 104 "KSC1"-like 1.1 x 103 5.6 x 103 5.5 x 104 “KSC2”-like 1.3 x 102 7.0 x 102 <102 moderate acidophiles
1.5 x 102 4.0 x 102 1.0 x 103
S-oxidizing bacteria* 2.5 x 102 1.0 x 103 <50 Heterotrophs (total) 50 2.1 x 105 1.6 x 104 NO-12 7.5 x 104 5.0 x 102 NO-13 5.1 x 104 3.0 x 103 NO-14 2.3 x 104 2.0 x 103 NO-15 1.4 x 104 5.0 x 103 NO-16 4.6 x 103 <102 NO-17 4.6 x 104 6.0 x 103
* Sulfur-oxidizing isolates which did not oxidize ferrous iron
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Isolate Nearest Relatives Identity (%)
NOen1
(AF376016)
Leptospirillum ferrooxidans DSM 2705T (X86776) 98.9
KSC1 (AF376017)
Acidithiobacillus ferrooxidans ATCC 23270T (AJ278718) 97.9
NO-8
(AF376018)
At. ferrooxidans ATCC 23270T 98.0
NO-25 (AF376019)
At. ferrooxidans ATCC 23270T 98.1
NO-37
(AF376020)
At. ferrooxidans ATCC 23270T 98.1
NO-12 (AF376021)
Acidocella facilis ATCC 35904T (D30774) 96.1
NO-13
(AF376022)
Acidiphilium rubrum ATCC 35905T (D30776) 99.6
NO-14 (AF376023)
A. cryptum ATCC 33463T (D30773) 99.8
NO-15
(AF376024)
Acidisphaera rubrifaciens strain HS-AP3T (D86512) 94.5
NO-16 (AF376025)
Frateuria aurantia DSM 6220T (AJ010481) 95.7
NO-17
(AF376026)
A. rubrum ATCC 35905T (D30776) 96.4
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Distribution of acidophilic heterotrophs in Kings Mine AMD
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Case study 2:Polymetallic Sulfide Bioleaching Pilot Plant:
Mintek, South Africa
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water & nutrients mineral concentrate
liquid pH adjustment & disposal
make-up tank
primary aeration tanks
secondary aeration tanks settling tank
solids to cyanidation & gold recovery
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TABLE 1. Conditions in the reactors of the pilot-scale biooxidation plant.
Reactor 1 Reactor 2 Reactor 3
pH
Cumulative residence time (days)
Soluble Cu (g/l)
Soluble Fe (g/l)*
Soluble Zn (g/l)
Sulfate (g/l)
1.6
3
17
13
6.5
65
1.5
4.5
19
14
7
67
1.3-1.4
6
20
15
7
70
*The iron was predominantly present as ferric iron.
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S. metallicus
Isolate MT16
Fp. acidiphilumT
L. ferrooxidansT
Isolate MT6
L. ferriphilumT
Sb. thermosulfidooxidansT
“Sb.yellowstonensis” y’sonensisyellowstonensis” YTF1 Sb. acidophilusT
Isolate NC
At. caldusT
Isolate MT1
Isolate MT17
“Fp. acidarmanus”
0.1
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Enrichment cultures:
• Select for target microorganisms
(e.g. thermophiles in low T samples)
• Allows detection and isolation of microorganisms present in relatively small numbers
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Enriching for Mesophilic Acidophiles
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Enriching for Mesophilic Acidophiles
Enrichment medium Streak to plate Enriches for
FeSO4 Feo At. ferrooxidans
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Enriching for Mesophilic Acidophiles
Enrichment medium Streak to plate Enriches for
FeSO4 Feo At. ferrooxidans
Fe2+/pyrite Feo Leptospirillum spp.
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Enriching for Mesophilic Acidophiles
Enrichment medium Streak to plate Enriches for
FeSO4 Feo At. ferrooxidans
Fe2+/pyrite Feo Leptospirillum spp.
S0 FeSo At. thiooxidans
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Enriching for Mesophilic Acidophiles
Enrichment medium Streak to plate Enriches for
FeSO4 Feo At. ferrooxidans
Fe2+/pyrite Feo Leptospirillum spp.
S0 FeSo At. thiooxidans
Fe2+/yeast extract Feo Ferrimicrobium spp.
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Enriching for Mesophilic Acidophiles
Enrichment medium Streak to plate Enriches for
FeSO4 Feo At. ferrooxidans
Fe2+/pyrite Feo Leptospirillum spp.
S0 FeSo At. thiooxidans
Fe2+/yeast extract Feo Ferrimicrobium spp.
Fe2+/yeast extract FeSo Sulfobacillus spp.
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Enriching for Mesophilic Acidophiles
Enrichment medium Streak to plate Enriches for
FeSO4 Feo At. ferrooxidans
Fe2+/pyrite Feo Leptospirillum spp.
S0 FeSo At. thiooxidans
Fe2+/yeast extract Feo Ferrimicrobium spp.
Fe2+/yeast extract FeSo Sulfobacillus spp.
Yeast extract YE3o Acidiphilium/Acidocella
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Enriching for Mesophilic Acidophiles
Enrichment medium Streak to plate Enriches for
FeSO4 Feo At. ferrooxidans
Fe2+/pyrite Feo Leptospirillum spp.
S0 FeSo At. thiooxidans
Fe2+/yeast extract Feo Ferrimicrobium spp.
Fe2+/yeast extract FeSo Sulfobacillus spp.
Yeast extract YE3o Acidiphilium/Acidocella
Yeast extract YE4o Acidobacterium/Acidisphaera
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Case study 3:Isolation of thermophilic acidophiles from sites in Yellowstone National Park, U.S.A.
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Frying Pan Hot Spring, Yellowstone N.P.
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Acidic site near Gibbon river, Yellowstone, U.S.A.
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Enrichment culture Ferrous sulfate/yeast extract Pyrite
YS1 Sulfobacillus-like (Y002) Sulfobacillus-like
YS2 Novel iron-oxidizers (Y005) Alicyclobacillus-like (Y004)
At. caldus-like
Novel iron-oxidizers (as Y005) Alicyclobacillus-like
At. caldus-like
YS3 No isolates obtained Sulfobacillus-like Gram negative heterotrophs (Y0013)
YS4 Alicyclobacillus-like Sulfobacillus-like
Gram negative heterotrophs (Y008)
Novel iron-oxidizers (asY005) Sulfobacillus-like
Gram negative heterotrophs (as Y008) At. caldus-like
YS5 Novel iron-oxidizer (as Y005) Sulfobacillus-like
Novel iron-oxidizers (as Y005) Sulfobacillus-like (Y0015, Y0016 & Y0017)
YS6 Alicyclobacillus-like Novel iron-oxidizers (as Y005) Gram negative heterotrophs (Y0012)
Sulfobacillus-like Acidimicrobium-like (Y0018)
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Acidophilic iron-oxidisers Acidophilic iron-reducers Acidophilic sulfur-oxidisers Acidophilic sulfate-reducers ?
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pHinternal 6.5
pHexternal 2.0CH3COOH
CH3COO- + H+
Acetic acid: CH3COOH CH3COO- + H+; pKa 4.75
(i.e., at pH 4.75, the dissociated and undissociated forms of the acid occur at equimolar concentrations).
pKa's of some other organic acids:
Lactic acid - 3.86Pyruvic acid - 2.50Formic acid - 3.75Citric acid - 3.68, 4.74 & 5.39
THE PROBLEM WITH ORGANIC ACIDS
(if you are an acidophile….)
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Overlay plate technique for
isolating and enumerating
acidophilic microorganisms
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Acidophilic Desulfosporosinus isolate
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Acidophilic Sulfidogenic Consortium
• Isolate “M1”
- A spore-forming acidophilic sulfate reducing bacterium (aSRB).
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• Isolate “M1”
- A spore-forming acidophilic sulfate reducing bacterium (aSRB).
- 94% 16S rRNA gene sequence identity to Desulfosporosinus orientis.
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• Isolate “M1”
- A spore-forming acidophilic sulfate reducing bacterium (aSRB).
- 94% 16S rRNA gene sequence identity to Desulfosporosinus orientis.
- Incomplete oxidizer of glycerol.
(4 glycerol + 3SO42- 4 acetic acid + 3H2S)
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Feedback inhibition of acetogenic SRB
in acidic liquors
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• Isolate “PFBC”
- A heterotrophic acidophilic Acidocella-like isolate.
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• Isolate “PFBC”
- A heterotrophic acidophilic Acidocella-like isolate.
- Isolated on solid medium, incubated anaerobically, from an supposedly pure SRB culture
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• Isolate “PFBC”
- A heterotrophic acidophilic Acidocella-like isolate.
- Isolated on solid medium, incubated anaerobically, from an supposedly pure SRB culture
- Grows on acetic acid aerobically.
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• Isolate “PFBC”
- A heterotrophic acidophilic Acidocella-like isolate.
- Isolated on solid medium, incubated anaerobically, from an supposedly pure SRB culture
- Grows on acetic acid aerobically.
- Does not grow in pure culture under anaerobic conditions
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Growth of M1 and PFBC in pure culture
+-Aerobic
--Anaerobic
Acetic acid
-+Anaerobic
--Aerobic
Glycerol
PFBCM1
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0
1
2
3
4
5
6
7
8
9
0 50 100 150Time (hours)
Anal
yte
(mM
)
SO4reduced
Glycerol
Aceticacid
Zn
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Hypothesis
• M1
4C3H8O3 + 3SO42- + 6H+
4CH3COOH + 3H2S + 4CO2 + 8H2O [1]
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Hypothesis
• M1
4C3H8O3 + 3SO42- + 6H+
4CH3COO- + 4H+ + 3HS- + 3H+ + 4CO2 + 8H2O [1]
• PFBC
4CH3COOH + 8H2O 8CO2 + 16H2 [2]
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Hypothesis
• M1
4C3H8O3 + 3SO42- + 6H+
4CH3COO- + 4H+ + 3HS- + 3H+ + 4CO2 + 8H2O [1]
• PFBC
4CH3COOH + 8H2O 8CO2 + 16H2 [2]
• M1
16H2 + 8H+ + 4SO42- 4H2S + 16H2O [3]
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Hypothetical scheme for anaerobic mixed culture
oxidation of glycerol
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• Overall reaction
4C3H8O3 + 7SO42- + 14H+
7H2S + 12CO2 + 16H2O [4]
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0
0.5
1
1.5
2
2.5
3
3.5
4
1 3 5 7 9Time (days)
Gly
cero
l and
sol
uble
Zn
(mM
)
GlycerolZn
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Mixed culture of Desulfosporosinus M1 and Acidocella PFBC:
a novel example of bacterial SYNTROPHY
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Preservation of acidophiles:
• Long term: low temperature freezing
(-70oC, in 7% dimethyl sulfoxide)
• Intermediate term: cold storage (4oC using “slow release” substrates
- coarse-grain pyrite for Fe-oxidizers
- elemental S for S-oxidizers
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