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

Identifi cation of isolates from physiological traits and/or sequence analysisof 16S rRN A genes

Isolation on solid media

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 ”

Identification of isolates from physiological traits and/or sequence analysisof 16S rRN A genes

Isolation on solid media

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 ”

Identification Identifi cation of isolates from physiological traits and/or sequence analysisof 16S rRN A genes

Isolation on solid media

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 ”

Identification of isolates isolates from Identifi cation of from physiologicaltraits traitsand/or and/orsequence sequence physiological analysisof 16S rRN genes analysis rRNAAgenes

Isolation on solid media

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 ”

Identification Identifi cation of isolates from physiological traits and/or sequence analysisof 16S rRN A genes

Isolation on solid media

PCR

T-RFLP analysis

16S rRNA genes

New peak(s) observed

Clone library constructed & sequenced

Probe design FISH analysis

:

Quantitative data

Identification of unknown prokaryotes

Modification/redesign of media for isolating “unculturables ”

Identification Identifi cation of isolates from physiological traits and/or sequence analysisof 16S rRN A genes

Isolation on solid media

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 ”

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 ironoxidizing 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

Iron-oxidizers

>103

Heterotrophs

80

10-4

200 8

10-5

0 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 tetrathionate

iron-oxidizers sulfur-oxidizers (heterotrophs)

FeTo

ferrous iron, (TSB) ~4.0 thiosulfate

moderately acidophilic Fe & Soxidizers 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

YE3o

yeast extract

~3.0

heterotrophs (extreme

yeast extract

~4.0

heterotrophs (moderate

acidophiles) YE4o acidophiles)

Target isolates

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) "KSC1"-like “KSC2”-like moderate acidophiles

1.4 x 103 1.1 x 103 1.3 x 102 1.5 x 102

6.7 x 103 5.6 x 103 7.0 x 102 4.0 x 102

5.6 x 104 5.5 x 104 <102 1.0 x 103

S-oxidizing bacteria*

2.5 x 102

1.0 x 103

<50

Heterotrophs (total) NO-12 NO-13 NO-14 NO-15 NO-16 NO-17

50

2.1 x 105 7.5 x 104 5.1 x 104 2.3 x 104 1.4 x 104 4.6 x 103 4.6 x 104

1.6 x 104 5.0 x 102 3.0 x 103 2.0 x 103 5.0 x 103 <102 6.0 x 103

* Sulfur-oxidizing isolates which did not oxidize ferrous iron

Isolate

Nearest Relatives

Identity (%)

NOen1 Leptospirillum ferrooxidans DSM 2705T (X86776) (AF376016)

98.9

KSC1 Acidithiobacillus ferrooxidans ATCC 23270T (AJ278718) (AF376017)

97.9

NO-8 At. ferrooxidans ATCC 23270T (AF376018)

98.0

NO-25 At. ferrooxidans ATCC 23270T (AF376019)

98.1

NO-37 At. ferrooxidans ATCC 23270T (AF376020)

98.1

NO-12 Acidocella facilis ATCC 35904T (D30774) (AF376021)

96.1

NO-13 Acidiphilium rubrum ATCC 35905T (D30776) (AF376022)

99.6

NO-14 A. cryptum ATCC 33463T (D30773) (AF376023)

99.8

NO-15 Acidisphaera rubrifaciens strain HS-AP3T (D86512) (AF376024)

94.5

NO-16 Frateuria aurantia DSM 6220T (AJ010481) (AF376025)

95.7

NO-17 A. rubrum ATCC 35905T (D30776) (AF376026)

96.4

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 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

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

pH

*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 ” y’sonensisyellowstone Sb. acidophilusT nsis” YTF1 Isolate NC At. caldusT 0.1

Isolate MT1

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 FeSO4

Streak to plate Feo

Enriches for At. ferrooxidans

Enriching for Mesophilic Acidophiles Enrichment medium

Streak to plate

FeSO4

Feo

Fe2+/pyrite

Feo

Enriches for At. ferrooxidans Leptospirillum spp.

Enriching for Mesophilic Acidophiles Enrichment medium

Streak to plate

FeSO4

Feo

Fe2+/pyrite

Feo

S0

FeSo

Enriches for At. ferrooxidans Leptospirillum spp. At. thiooxidans

Enriching for Mesophilic Acidophiles Enrichment medium

Streak to plate

FeSO4

Feo

Fe2+/pyrite

Feo

S0

FeSo

Fe2+/yeast extract

Feo

Enriches for At. ferrooxidans Leptospirillum spp. At. thiooxidans Ferrimicrobium spp.

Enriching for Mesophilic Acidophiles Enrichment medium

Streak to plate

FeSO4

Feo

Fe2+/pyrite

Feo

S0

FeSo

Fe2+/yeast extract

Feo

Fe2+/yeast extract

FeSo

Enriches for At. ferrooxidans Leptospirillum spp. At. thiooxidans Ferrimicrobium spp. Sulfobacillus spp.

Enriching for Mesophilic Acidophiles Enrichment medium

Streak to plate

Enriches for

FeSO4

Feo

At. ferrooxidans

Fe2+/pyrite

Feo

S0

FeSo

Fe2+/yeast extract

Feo

Fe2+/yeast extract

FeSo

Sulfobacillus spp.

Yeast extract

YE3o

Acidiphilium/Acidocella

Leptospirillum spp. At. thiooxidans Ferrimicrobium spp.

Enriching for Mesophilic Acidophiles Enrichment medium

Streak to plate

Enriches for

FeSO4

Feo

At. ferrooxidans

Fe2+/pyrite

Feo

S0

FeSo

Fe2+/yeast extract

Feo

Fe2+/yeast extract

FeSo

Sulfobacillus spp.

Yeast extract

YE3o

Acidiphilium/Acidocella

Yeast extract

YE4o

Acidobacterium/Acidisphaera

Leptospirillum spp. At. thiooxidans Ferrimicrobium spp.

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-oxidizer (as Y005) Sulfobacillus-like Alicyclobacillus-like

Novel iron-oxidizers (asY005) Sulfobacillus-like Gram negative heterotrophs (as Y008) At. caldus-like Novel iron-oxidizers (as Y005) Sulfobacillus-like (Y0015, Y0016 & Y0017) Novel iron-oxidizers (as Y005) Gram negative heterotrophs (Y0012) Sulfobacillus-like Acidimicrobium-like (Y0018)

YS5 YS6

Acidophilic iron-oxidisers √ Acidophilic iron-reducers √ Acidophilic sulfur-oxidisers √ Acidophilic sulfate-reducers ?

THE PROBLEM WITH ORGANIC ACIDS (if you are an acidophile….)

CH3COO- + H+

pHinternal 6.5

pHexternal 2.0

Acetic acid: CH3COOH

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 Acidocellalike isolate.

• Isolate “PFBC” - A heterotrophic acidophilic Acidocellalike isolate. - Isolated on solid medium, incubated anaerobically, from an supposedly pure SRB culture

• Isolate “PFBC” - A heterotrophic acidophilic Acidocellalike isolate. - Isolated on solid medium, incubated anaerobically, from an supposedly pure SRB culture - Grows on acetic acid aerobically.

• Isolate “PFBC” - A heterotrophic acidophilic Acidocellalike 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

Analyte (mM)

7 6

SO4 reduced

5

Glycerol

4 Acetic acid

3 2

Zn

1 0 0

50

100 Time (hours)

150

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 Glycerol and soluble Zn (mM)

3.5 3 2.5

Glycerol Zn

2 1.5 1 0.5 0 1

3

5 Time (days)

7

9

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