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”?
Culture-dependent
methods
• enumeration
• plate isolation
• enrichment cultures
• micromanipulation
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
solid media

Clone library


Identification
Isolation on
solid media

Clone library


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

Clone library


Identification
Isolation on
solid media

Clone library
Probe design Quantitative

: Identification of
FISH analysis data unknown prokaryotes

Identification
Isolation on
solid media

Clone library


Enumeration of microorganisms:
• Direct counts (phase contast microscopy;

Thoma cell)
• Direct counts (stained cells)
• Most probable number (MPN) counts
• Plate counts
• 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

Thoma cell)


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

Thoma cell)


Thoma cell)
• Plate counts
• 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
solid media

• Purity of the gelling agent (e.g. agar)

solid media


→ wash agar before sterilization
solid media

• Hydrolysis of the gelling agent

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
heterotrophs
• Extremely acidic environments are mostly

oligotrophic (contain little organic C)
heterotrophs

• acidophilic heterotrophs (like autotrophs)

may be inhibited by medium-high
concentrations of dissolved carbon, and
very small amounts of organic acids
heterotrophs

• 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
• Restricted metabolic capabilities

• catabolizes organic acids (primary

inhibitory compounds in solid media)
• 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)
(AF376019)
(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
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 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).

- 94% 16S rRNA gene sequence identity to
Desulfosporosinus orientis.
- 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.

like isolate.
- Isolated on solid medium, incubated
anaerobically, from an supposedly
pure SRB culture

like isolate.
pure SRB culture
- Grows on acetic acid aerobically.

like isolate.
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

Approaches and Techniques For Isolating and Cultivating Acidophiles

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Approaches and Techniques for

Isolating and Cultivating Acidophiles

School of Biological Sciences,

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

Probe design: Identification of

Modification/redesign of media for

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

Probe design: Identification of

Modification/redesign of media for

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

Probe design: Identification of

Modification/redesign of media for

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

Probe design: Identification of

Modification/redesign of media for

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

Probe design Quantitative

Modification/redesign of media for

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

Probe design: Identification of

Modification/redesign of media for

• Direct counts (phase contast microscopy;

• Direct counts (stained cells)

• Most probable number (MPN) counts

• Direct counts (phase contast microscopy; Thoma

• Direct counts (phase contast microscopy;

• Direct counts (stained cells)

• Direct counts (stained cells)

• Direct counts (phase contast microscopy;

• Direct counts (stained cells)

• Most probable number (MPN) counts

• Direct counts (phase contast microscopy;

• Direct counts (stained cells)

• Most probable number (MPN) counts

• Sensitivity of many acidophiles to organic

• Sensitivity of many acidophiles to organic

• Purity of the gelling agent (e.g. agar)

• Sensitivity of many acidophiles to organic

• Purity of the gelling agent (e.g. agar)

• Sensitivity of many acidophiles to organic

• Purity of the gelling agent (e.g. agar)

• Hydrolysis of the gelling agent

• Hydrolysis of the gelling agent

→ need for continuous removal of small

• Contains both ferrous iron and tryptone

• Designed to promote the growth of iron-

Iron-oxidizers >103 200 0

Code Energy sources pH Target isolates

Feo ferrous iron/(TSB) ~2.6 iron-oxidizers

FeSo ferrous iron, (TSB) ~2.6 iron-oxidizers

FeTo ferrous iron, (TSB) ~4.0 moderately acido-

• Extremely acidic environments are mostly

• Extremely acidic environments are mostly

• acidophilic heterotrophs (like autotrophs)

• Extremely acidic environments are mostly

• acidophilic heterotrophs (like autotrophs)

• overlay media again produce higher

• Restricted metabolic capabilities

• Restricted metabolic capabilities

• catabolizes organic acids (primary

• Restricted metabolic capabilities

• catabolizes organic acids (primary