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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
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 Culture-independent
methods methods
• PCR-dependent
approaches
• enumeration
- clone libraries
• plate isolation
- T-RFLP, DGGE etc.
• enrichment cultures
• PCR-independent
• micromanipulation
approaches
- FISH
- flow cytometry
Identifi cation of isolates from
Isolation on
physiological traits and/or sequence
solid media
analysisof 16S rRN A genes

PCR 16S rRNA T-RFLP New peak(s)


genes analysis observed

Clone library
constructed & sequenced

Probe design: Identification of


FISH analysis unknown prokaryotes

Modification/redesign of media for


isolating “unculturables ”
Identification of isolates from
Isolation on
physiological traits and/or sequence
solid media
analysisof 16S rRN A genes

PCR 16S rRNA T-RFLP New peak(s)


genes analysis observed

Clone library
constructed & sequenced

Probe design: Identification of


FISH analysis unknown prokaryotes

Modification/redesign of media for


isolating “unculturables ”
Identification
Identifi cation of isolates from
Isolation on
physiological traits and/or sequence
solid media
analysisof 16S rRN A genes

PCR 16S rRNA T-RFLP New peak(s)


genes analysis observed

Clone library
constructed & sequenced

Probe design: Identification of


FISH analysis unknown prokaryotes

Modification/redesign of media for


isolating “unculturables ”
Identification
Identifi of isolates
cation of isolates from
from
Isolation on
physiologicaltraits
physiological traitsand/or
and/orsequence
sequence
solid media
analysisof 16S rRN
analysis rRNAAgenes
genes

PCR 16S rRNA T-RFLP New peak(s)


genes analysis observed

Clone library
constructed & sequenced

Probe design: Identification of


FISH analysis unknown prokaryotes

Modification/redesign of media for


isolating “unculturables ”
Identification
Identifi cation of isolates from
Isolation on
physiological traits and/or sequence
solid media
analysisof 16S rRN A genes

PCR 16S rRNA T-RFLP New peak(s)


genes analysis observed

Clone library
constructed & sequenced

Probe design Quantitative


: Identification of
FISH analysis data unknown prokaryotes

Modification/redesign of media for


isolating “unculturables ”
Identification
Identifi cation of isolates from
Isolation on
physiological traits and/or sequence
solid media
analysisof 16S rRN A genes

PCR 16S rRNA T-RFLP New peak(s)


genes analysis observed

Clone library
constructed & sequenced

Probe design: Identification of


FISH analysis unknown prokaryotes

Modification/redesign of media for


isolating “unculturables ”
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

Acidiphilium sp.

At. ferrooxidans
FeTSB medium: typical data where numbers
of iron-oxidizers > acidophilic heterotrophs

Dilution
Colonies nos. 10-3 10-4 10-5

Iron-oxidizers >103 200 0


Heterotrophs 80 8 0
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. ferrooxidans

At. thiooxidans

Ferrimicrobium
Colonies of moderate acidophiles: FeTo medium

Thiomonas sp

S-oxidizer
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.

Acidobacterium sp.

A.rubrum

Fratauria sp.

Acidiphilium 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 Leptospirillum ferrooxidans DSM 2705T (X86776) 98.9
(AF376016)
KSC1 Acidithiobacillus ferrooxidans ATCC 23270T (AJ278718) 97.9
(AF376017)
NO-8 At. ferrooxidans ATCC 23270T 98.0
(AF376018)
NO-25 At. ferrooxidans ATCC 23270T 98.1
(AF376019)
NO-37 At. ferrooxidans ATCC 23270T 98.1
(AF376020)
NO-12 Acidocella facilis ATCC 35904T (D30774) 96.1
(AF376021)
NO-13 Acidiphilium rubrum ATCC 35905T (D30776) 99.6
(AF376022)
NO-14 A. cryptum ATCC 33463T (D30773) 99.8
(AF376023)
NO-15 Acidisphaera rubrifaciens strain HS-AP3T (D86512) 94.5
(AF376024)
NO-16 Frateuria aurantia DSM 6220T (AJ010481) 95.7
(AF376025)
NO-17 A. rubrum ATCC 35905T (D30776) 96.4
(AF376026)
Distribution of acidophilic heterotrophs in Kings
Mine AMD
Case study 2:
Polymetallic Sulfide Bioleaching Pilot Plant:
Mintek, South Africa
mineral
concentrate water & nutrients

liquid pH
adjustment &
disposal

make-up tank

primary secondary aeration


aeration tanks 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 1.6 1.5 1.3-1.4


Cumulative residence time (days) 3 4.5 6
Soluble Cu (g/l) 17 19 20
Soluble Fe (g/l)* 13 14 15
Soluble Zn (g/l) 6.5 7 7
Sulfate (g/l) 65 67 70

*The iron was predominantly present as ferric iron.


S. metallicus

Isolate MT16

Fp. acidiphilumT

Isolate MT17

“Fp. acidarmanus”

L. ferrooxidansT

Isolate MT6

L. ferriphilumT

Sb. thermosulfidooxidansT

“Sb.yellowstonensis

Sb. acidophilusT
y’sonensisyellowstone
nsis” YTF1
Isolate NC

At. caldusT

Isolate MT1
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) Novel iron-oxidizers (as Y005)


Alicyclobacillus-like (Y004) Alicyclobacillus-like
At. caldus-like At. caldus-like

YS3 No isolates obtained Sulfobacillus-like


Gram negative heterotrophs (Y0013)

YS4 Alicyclobacillus-like Novel iron-oxidizers (asY005)


Sulfobacillus-like Sulfobacillus-like
Gram negative heterotrophs Gram negative heterotrophs (as Y008)
(Y008) At. caldus-like
YS5 Novel iron-oxidizer (as Y005) Novel iron-oxidizers (as Y005)
Sulfobacillus-like 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 ?
THE PROBLEM WITH ORGANIC ACIDS
(if you are an acidophile….)

pHinternal 6.5 CH3COO- + H+

pHexternal 2.0
CH3COOH

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 - 3.86


Pyruvic acid - 2.50
Formic acid - 3.75
Citric acid - 3.68, 4.74 & 5.39
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
M1 PFBC
Glycerol
Aerobic - -
Anaerobic + -
Acetic acid
Aerobic - +
Anaerobic - -
9
8
7
SO4
6 reduced
Analyte (mM)

5 Glycerol
4
3 Acetic
acid
2
Zn
1
0
0 50 100 150
Time (hours)
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]
4
3.5
Glycerol and soluble Zn (mM)

3
2.5
Glycerol
2 Zn
1.5
1
0.5
0
1 3 5 7 9
Time (days)
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