enrichment of black yeasts under atmospheres of aromatic

Francesc Prenafeta Boldú

Enrichment of black yeastsunder atmospheres of aromatichydrocarbons:Impact on biotechnologicalapplications

Centre impulsat per: Centre adscrit a:

Halophilic fungi in hypersaline salternsSelbmann Stud Mycol 61:1 (2008)

…how extreme is extreme for black yeasts?

Cryptoendolitic fungi in the Antarctic Dry ValleysRuisi et al. Rev Environ Sci Biotech 6 (2006)

Radiotrophic fungi in ChernobilDadachova et al. PLoS One 2 (2007) Fungi in creosoted wood

Zhao et al. Microb Ecol (2010)

Introduction

Isolation from the environment

Zhao et al. Microb Ecol (2010)

Introduction

A lab biofilter treating toluene became fully colonized with a dark fungus, then identified as a Cladosporium sphaerospermum(the correct identity is Cladophialophora saturnica)

This biofilter displayed a very robust and stable performance, despite poor irrigation and relatively high organic loads.

Fungal growth on toluene as sole carbon and energy source was demonstrated

Fungal biofiltration

Weber et al. Appl Environ Microbiol 61 (1995)

“Hydrocarbonophilic” black yeasts

Gas biofiltration techniques are characterized as:

-Relatively inexpensive solution for treating gases polluted with a low VOC’s content

-More sustainable technology than physicochemical alternative processes

-Easy operation and maintenance

-Tendency to clog due to excessive biomass growth or packing deterioration

Effluent (clean air)

Influent (air polluted with VOC’s)

Enrichment of microorganismsgrowing on VOC’s

Active biomass is exposed to:

-Fluctuating concentrations of toxic chemicals

-Dry conditions

-Low nutrient content

-Acidic conditions



Benzene Toluene Ethylbenzene

o-Xylene m-Xylene p-Xylene

Bulk chemicals used in the synthesis of several products (pesticides, explosives, etc.) and materials (plastics), used as solvents (painting and coating), as fuel additives, etc.

Properties of BTEX hydrocarbons:

-Volatile chemicals (atmospheric pollutants)

-Relatively soluble (water pollutants)

-Toxic to men and the environment

-Easily biodegradable under aerobic conditions

BTEX monoaromatic hydrocarbons


Treatment of styrene-containing varnishing emissions(www.lm-mibi.uni-bonn.de)

Treatment of solvent vapors of paints in a car manufacturing plant(Daimler-Benz)

Full-scale biofiltration of volatile aromatic hydrocarbons



Bioventing of BTEX polluted soil due to leakage of underground storage tanks of fuels and petroleum derivates (i.e. former gasoline stations)



Black yeasts in hydrocarbon-rich environments

Kerosene fungus (Amorphoteca resinae)

Interest in this fungus arised by reports of its occurrence in storage and aircraft fuel tanks containing aviation fuel in the early 1960's

Sheridan et al. TUATARA 19 (1971)


Fungal metabolism of hydrocarbons

Assimilative metabolism of aliphatics and phenols

Different black-yeasts in the Dothideales assimilate aliphatic hydrocarbons and phenolic compounds (i.e. Amorphoteca resinae, Aureobasidium pullulans, Cladosporium sphaerospermum, etc.)

Co-metabolic degradation of aromatic hydrocarbons

Biodegradation of BTEX in a biofilters inoculated with the white-rot fungus Phanerochaete chrysosporium.

Substrates were not used as carbon and energy sources, and biofilters were supplemented with strawBraun-Lüllemann et al. Battelle Press (1995)

Assimilation of aromatic hydrocarbons

Several species of the Chaetothyriales are able to grow on BTEX hydrocarbons as the sole source of carbon and energyPrenafeta et al. FEMS Microbiol Rev 30 (2006)


Phylogeny of black yeasts

EukaryotaFungi/Metazoa groupFungiDikaryaAscomycotaPezizomycotinaEurotiomycetesChaetothyriomycetidaeChaetothyrialesHerpotrichiellaceae

…DothideomycetesDothideomycetidaeDothideales

SSU rDNA


Are air biofilters treating aromatic hydrocarbons actually breeding for pathogenic fungi?

BSL-2 Toluene-degrading bacteria:

Pseudomonas putidaPseudomonas aeruginosaMycobacterium vacaeMycovacterium spp.Nocardia spp.

Potential biohazard of fungal biofilters


Specific intron (558 bp) at position 1768 in the SSU rDNA

Badali et al. (submitted)

10%

CBS 650.93 T (brain)

CBS 556.83 T (rotten wood)

CBS 899.68 T (subcutaneous)

CBS 579.76 (brain)

CBS 110553 (hydrocarbon-polluted soil)

CBS 637.69 (polyvinyl alcohol solution)

CBS 116.97 (hydrocarbon-polluted soil)

CBS 101158 (brain)

CBS 633.69 (railway tie)

CBS 271.37 T (mycosis)

CBS 126.86 T (cutaneous)

CBS 982.96 (soil)

CBS 113408 (biofilter)

DH 13236 (hydrocarbon-polluted soil)

DAOM 216391 (galleries of bark beetles in pine)

CBS 317.33 T (pine wood)

CBS 160.54T (cutaneous)

CBS 269.37 T (chromomycosis)

CBS 814.95 (biofilter)

CBS 109154 (brain)

CBS 102586 (brain)

CBS 306.94 T (subcutaneous)CBS 102461 (brain)CBS 147.84 T (subcutaneous)

CBS 110551 (biofilter)CBS 102230 (plant debris)CBS 114326 (biofilter)

CBS 102225 (rotten wood)CBS 115830 (brain)

CBS 979.96 T (subcutaneous)DH 13029 (brain)

CBS 155.53 (brain)CBS 173.52 T (brain)

CBS 859.96 (plant debris)CBS 101252 (brain)

CBS 123.37 T (symbiont lice)CBS 102400 (biofilter)

DH 13725 (railway tie)CBS 207.35 T (chromomycosis)

CBS 232.33 T (wood pulp)CBS 117.97 (hydrocarbon-polluted soil)

CBS 642.82 (rotten wood)DH 11807 (railway tie)

CBS 115831 (browncoal)CBS 110555 (hydrocarbon-polluted soil)

CBS 528.76 (cutaneous)CBS 507.90 T (mycetoma)

CBS 111763 T (foot lesion)DH 13054 (brain)

CBS 680.76 (activated sludge)CBS 725.88 T (subcutaneous)

CBS 243.85 T (spruce resin)CBS 238.93 (biofilter)

100

99

100

91

86

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97

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93

100

95

100

Cladophialophora arxiiCladophialophora devriesii

Fonsecaea pedrosoi

Fonsecaea monophora

Cladophialophora emonsii

Cladophialophora bantiana

Cladophialophora psammophila sp. nov.

Cladophialophora boppiCladophialophora minourae

Cladophialophora carrionii

Exophiala lecanii-corniRhinocladiella atrovirens

Exophiala dermatitidis

Exophiala heteromorpha

Ramichloridium mackenziei

Exophiala xenobiotica sp. nov.

Exophiala oligosperma

Rhinocladiella similis

Exophiala spiniferaExophiala jeanselmei

Phialophora sessilis (Outgroup)

Cladophialophora saturnica sp. nov.Cladophialophora immunda sp. nov.

Natural environments-Soil-Wood/resin-Plant debris-Rotten wood-Bark insect galeries

Artificial environments-Oil contaminated soil-Creosoted wood-Wood pulp-Browncoal-Activated sludge

Human pathogens-Cutaneous-Subcutaneous-Neurotropic

Neighbor JoiningITS1-5.8S-ITS2

Prenafeta et al. FEMS Microbiol Rev 30 (2006)

Natural sources of monoaromatic hydrocarbons


Myroxylonbalsamum (Fabales)

tolu tree

Styrax benzoin(Ericales)

styrax

J. Buckingham, et al. (1995) Dict natur Prod

Helianthus annuus(Asterales)sunflower

Pinus sylvestris(Coniferales)

scots pine

A.C. Heiden et al. (1999) Geophys Res Let

Quercus ilex(Fagales)

evergreen oak

R. Holzinger et al. (2000) J. Geophys. Res. Atmosph.P.Vrkocova et al. (2000) Biochem. System. Ecol.

Quercus robur(Fagales)

oak

Ips pini(Coleoptera)

pine engraver beetle

Gries, G. et al. (1990) Experientia

Salt solution(for humidity control)

Perlite soaked with mineral mediumVolatile AH

(via de gas phase)

Solid state-like enrichment on BTEX hydrocarbons

Selective isolation of VOC-degrading black yeasts

Prenafeta et al. Mycol Res 105 (2001)

Isolation of C. psammophila CBS 110553 on toluene

from a BTEX polluted soil treated by bioventing


0

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0 5 10 15Time (d)

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¡To

luen

e (µ

mol

) ¨C

O2 (µm

ol)

1

10

100

1000

6 8 10 12(d)

(µm

ol)

Growth on toluene of C. psammophila CBS 110553

C-toluene recovery as CO2 > 80%


Fungal metabolism of toluene

Metabolic pathway for the fungal assimilation of toluenePrenafeta et al. Appl Environ Microbiol 67 (2001)


Fungal metabolism of toluene

Sources of inocula:

-Soil polluted with PAH’s

-BTEX Biofilter biomass

-Creosoted wood


Substrate:

-Benzene

Fungal metabolism of benzene

Mineral medium Mineral medium

+ benzene

Mineral medium +

benzene + glucose

Solid state-like incubations of railway ties (cresoted wood) under atmospheres of benzene yielded several strains of two black yeast species:

-Exophiala xenobiotica

-Exophiala bergeri


Fungal metabolism of benzene

Operational parameters:

-Modular configuration-Organic packing-Poor irrigation (20-30% humidity)-Increasing organic load (5-70 g h-1 m-3)

Hydrocarbon degrading black yeasts in biofilters

Fungal-bacterial interactions in the biofiltration of BTEX


Organic Loading Rate (g h

-1m-3)Re

mov

al E

ffic

ienc

y (%

)

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EBRT = 188 sCi = 55-473 g m-3

EBRT = 94 sCi = 93-238 g m-3

EBRT = 47 sCi = 29-247 g m-3

Ethylbenzene

EBRT = 188 sCi = 44-369 g m-3

EBRT = 94 sCi = 51-187 g m-3

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P-Xylene

Time of operation (days)

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EBRT = 142 sCi = 27-422 g m-3

EBRT = 87 sCi = 87-312 g m-3

EBRT = 44 sCi = 184-265 g m-3

Toluene

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Time of operation (days)

Pack

ingm

oisu

tre

(%)

Module M2

Module M1


Colonization of the biofilter packing with fungi

Packing material at time 0 days Packing material at time 190 days

SEM imaging of the biofilter biomass

Bacterial-fungal interactions in biofilters for BTEX removal

Co-operation with the “Universidad del País Vasco”

Alcaligenes

Alcanivorax dieselolei

Microbacteriumtrichotecenolyticum

Bacteriodetes

Rhodococus

Streptomyces albiaxialis

Nocardiodes

Flexibacteraceae

Bacteriodetes

Odyssella

Deinococcus

Actinomadura

Gemmatimonadetes Thermomicrobium

Chelatobacter

Sanguibacter

Streptomyces

T E p-X p-X PackingM1 M1 M1 M2

Exophiala

Exophialaoligosperma

Aspergillus versicolor

Fusarium solani

Aspergillus giganteus

T E p-X p-X PackingM1 M1 M1 M2

Bacterial 16S rDNAF340(GC)-R907 primers

Fungal ITS1 rDNAITS1F(GC)-ITS2 primers

Molecular community profiling (PCR-DGGE)

Fungal versus bacterial microbial community structure


Nr of copies of bacterial 16S and fungal ITS rDNA genes (qPCR)

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1.E+04

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1.E+07

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1.E+10

1.E+11

1.E+12

1.E+13

1.E+14

Toluene Ethylbenzene Xylene Packing Inoculum

Fungal/Bacterial ratio

Nrof

rRNA

ene

copi

es g

-1

Fungal-bacterial biomass ratio


Conclusions

The ecology presence of sibling species with a very divergent ecology (pathogenic versus xenobiont) suggest that black yeasts are experimenting an intense process of speciation in recent evolution

Virulence and capacity to assimilate aromatic hydrocarbons is predominant within the Herpotrichiellaceae family (Chaetotyriales), while members of the Dothideales lack those characteristics

The amazing capacities of black yeast potentially have several biotechnological applications that are currently investigated: production of extremetolerant enzymes, bioremediation agents (biodegradation of aromatic compounds), etc.

Acknowledgements

http://www.blackyeast.org/

Ana ElíasAstrid Barona

Sybren de HoogHamid BadaliJingjun Zhao

CBS Centraalbureau voor Schimmelcultures

Marc ViñasMíriam Guivernau

enrichment of black yeasts under atmospheres of aromatic

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