Approaches and Techniques for Isolating and Cultivating Acidophiles
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Transcript of Approaches and Techniques for Isolating and Cultivating Acidophiles
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
What methods can (and should) be used to study “mine microbiology”?
What methods can (and should) be used to study “mine microbiology”?
Culture-dependent methods
• enumeration• plate isolation• enrichment cultures• micromanipulation
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
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
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
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
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
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
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
Enumeration of microorganisms:
• Direct counts (phase contast microscopy; Thoma cell)
• Direct counts (stained cells)
• Most probable number (MPN) counts
• Plate counts
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
Enumeration of microorganisms:
• Direct counts (phase contast microscopy; Thoma cell)
• Direct counts (stained cells)
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
Trefriw biofilm stained with DAPI
Enumeration of microorganisms:
• Direct counts (phase contast microscopy; Thoma cell)
• Direct counts (stained cells)
• Most probable number (MPN) counts
Enumeration of microorganisms:
• Direct counts (phase contast microscopy; Thoma cell)
• Direct counts (stained cells)
• Most probable number (MPN) counts
• Plate counts
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
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
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)
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
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
Problems with growing acidophiles on solid media
• Hydrolysis of the gelling agent
need for continuous removal of small molecular weight hydrolysates
Early plate formulation: “FeTSB” medium
• Contains both ferrous iron and tryptone soya broth
• Designed to promote the growth of iron-oxidizing and heterotrophic acidophiles
Acidophilic colonies: FeTSB medium
At. ferrooxidans
Acidiphilium sp.
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
Overlay plate technique for
isolating and enumerating
acidophilic microorganisms
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
Acidophilic colonies: FeSo medium
At. thiooxidans
Ferrimicrobium
At. ferrooxidans
Colonies of moderate acidophiles: FeTo medium
S-oxidizer
Thiomonas sp
Isolation/enumeration of acidophilic heterotrophs
Isolation/enumeration of acidophilic heterotrophs
• Extremely acidic environments are mostly oligotrophic (contain little organic C)
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
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
Underlay heterotroph: Acidocella WJB3
Underlay heterotroph: Acidocella WJB3
• Restricted metabolic capabilities
Underlay heterotroph: Acidocella WJB3
• Restricted metabolic capabilities
• catabolizes organic acids (primary inhibitory compounds in solid media)
Underlay heterotroph: Acidocella WJB3
• Restricted metabolic capabilities
• catabolizes organic acids (primary inhibitory compounds in solid media)
• does not grow on yeast extract or glycerol
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)
Colonies of heterotrophic acidophiles: YE3o medium
Thiomonas s
Case study 1: Roeros copper mine, Norway
Roeros copper mine, Norway
Acidophilic iron-oxidizers: Roeros copper mine, Norway
Acidophilic heterotrophs: Roeros copper mine, Norway
Acidocella sp.
A.rubrum
Acidiphilium sp.
Acidobacterium sp.
Fratauria sp.
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
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
Distribution of acidophilic heterotrophs in Kings Mine AMD
Case study 2:Polymetallic Sulfide Bioleaching Pilot Plant:
Mintek, South Africa
water & nutrients mineral concentrate
liquid pH adjustment & disposal
make-up tank
primary aeration tanks
secondary aeration tanks settling tank
solids to cyanidation & gold recovery
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.
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
Enrichment cultures:
• Select for target microorganisms
(e.g. thermophiles in low T samples)
• Allows detection and isolation of microorganisms present in relatively small numbers
Enriching for Mesophilic Acidophiles
Enriching for Mesophilic Acidophiles
Enrichment medium Streak to plate Enriches for
FeSO4 Feo At. ferrooxidans
Enriching for Mesophilic Acidophiles
Enrichment medium Streak to plate Enriches for
FeSO4 Feo At. ferrooxidans
Fe2+/pyrite Feo Leptospirillum spp.
Enriching for Mesophilic Acidophiles
Enrichment medium Streak to plate Enriches for
FeSO4 Feo At. ferrooxidans
Fe2+/pyrite Feo Leptospirillum spp.
S0 FeSo At. thiooxidans
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.
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.
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
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
Case study 3:Isolation of thermophilic acidophiles from sites in Yellowstone National Park, U.S.A.
Frying Pan Hot Spring, Yellowstone N.P.
Acidic site near Gibbon river, Yellowstone, U.S.A.
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)
Acidophilic iron-oxidisers Acidophilic iron-reducers Acidophilic sulfur-oxidisers Acidophilic sulfate-reducers ?
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….)
Overlay plate technique for
isolating and enumerating
acidophilic microorganisms
Acidophilic Desulfosporosinus isolate
Acidophilic Sulfidogenic Consortium
• Isolate “M1”
- A spore-forming acidophilic sulfate reducing bacterium (aSRB).
• Isolate “M1”
- A spore-forming acidophilic sulfate reducing bacterium (aSRB).
- 94% 16S rRNA gene sequence identity to Desulfosporosinus orientis.
• 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)
Feedback inhibition of acetogenic SRB
in acidic liquors
• Isolate “PFBC”
- A heterotrophic acidophilic Acidocella-like isolate.
• Isolate “PFBC”
- A heterotrophic acidophilic Acidocella-like isolate.
- Isolated on solid medium, incubated anaerobically, from an supposedly pure SRB culture
• 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.
• 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
Growth of M1 and PFBC in pure culture
+-Aerobic
--Anaerobic
Acetic acid
-+Anaerobic
--Aerobic
Glycerol
PFBCM1
0
1
2
3
4
5
6
7
8
9
0 50 100 150Time (hours)
Anal
yte
(mM
)
SO4reduced
Glycerol
Aceticacid
Zn
Hypothesis
• M1
4C3H8O3 + 3SO42- + 6H+
4CH3COOH + 3H2S + 4CO2 + 8H2O [1]
Hypothesis
• M1
4C3H8O3 + 3SO42- + 6H+
4CH3COO- + 4H+ + 3HS- + 3H+ + 4CO2 + 8H2O [1]
• PFBC
4CH3COOH + 8H2O 8CO2 + 16H2 [2]
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]
Hypothetical scheme for anaerobic mixed culture
oxidation of glycerol
• Overall reaction
4C3H8O3 + 7SO42- + 14H+
7H2S + 12CO2 + 16H2O [4]
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
Mixed culture of Desulfosporosinus M1 and Acidocella PFBC:
a novel example of bacterial SYNTROPHY
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