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