Scientific Highlight April 2010

co-ordinated with the Director of the Institute

Institute Institute of Groundwater Ecology

PSP-Element: G-504300-002

Person to contact for further enquiries: Dr. Tillmann Lüders, [email protected], Tel. 3687

Title of the Highlight: DNA-SIP identifies sulfate-reducing as important toluene-degraders in tar-oil-contaminated aquifer sediment

Keywords: Groundwater resources, ecosystem services, natural attenuation, BTEX, microbial key-players

Central statement of the Highlight in one sentence: We have proven that uncultured members of the Clostridia, classically considered as fermenters and often also pathogenic microbes, are responsible for toluene degradation under sulfate-reduction in a contaminated aquifer.

Text of the Highlight: Groundwater is the most important drinking water resources of our society. Still, global groundwater resources are constantly challenged by a multitude of contaminants such as aromatic hydrocarbons. Especially in anaerobic habitats, a large diversity of unrecognized microbes is hypothesized to be responsible for their degradation. However, the true identity of these populations and the factors that control their activities in situ are still poorly understood.

Here, by using innovative 13C-labelling technologies for DNA, we have identified the most active sulfate-reducing toluene degraders within a diverse aquifer microbial community from a former Gasworks site in Germany. Surprisingly, the identified key-players were related to spp. within the (Clostridia). Up to now, members of the Clostridia have not been recognized as important sulfate-reducing contaminant degraders, rather they are classically considered as fermenters and often also pathogenic microbes. Also, carbon flow from the contaminant into degraders was unexpectedly low, pointing toward high ratios of heterotrophic CO2-fixation during assimilation of acetyl-CoA

1 from toluene, which may represent an important and unrecognized ecophysiological constraint for these degraders.

The functional identification of these novel degraders, along with their affiliated, previously unidentified degradation (bssA) genes fosters our understanding of the biodiversity and ecophysiology of relevant microbial key players in natural attenuation. Thus, these results contribute to a better understanding of groundwater ecosystem services and pave the way towards novel monitoring and management concepts for contaminated groundwater.

Publication: Winderl, C., Penning, H., von Netzer, F., Meckenstock, R.U., and Lueders, T. (2010) DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil contaminated aquifer sediment. ISME J (in press).

Taking account of the HMGU mission: We investigate an ecosystem with essential significance for human health. Groundwater ecosystems deliver important services to our society, whereas the contamination of ground- and drinking water by carcinogenic BTEX components is of major concern. The results of this project funded by DFG are an essential contribution to the sustainable of groundwater resources and to drinking water protection.

The internal HMGU co-operation partners with whom the Highlight was compiled, if appropriate: G-504300-004 G-550700-001 S-779310-5043-001 S-700015-5043-001

2 Sulfate-reducingSulfate-reducing ClostridiaClostridia areare importantimportant toluene-degraderstoluene-degraders inin contaminatedcontaminated aquifersaquifers Institute of Groundwater EInstitutcology

Contaminant degradation in groundwater ecosystems

 Which microbes are really relevant? Sampling  What is controlling their activity? Oil

Plume Degradation ?

Groundwater ? StableStable isotopeisotope labellinglabelling toto identifyidentify microbialmicrobial key-playerskey-players Institute of Groundwater EInstitutcology

Detection of 13C-labelled DNA and rRNA by “Stable Isotope Probing” DensityDensity gradientsgradients ofof aquiferaquifer sedimentsediment DNADNA afterafter 1313 C-tolueneC-toluene consumptionconsumption Institute of Groundwater EInstitutcology

Bacterial rRNA gene abundance [ratio of max] 12 13 0.0 0.2 0.4 0.6 0.8 1.0 1.2 C-Toluene C-Toluene 1.66

1.68 “light” DNA

1.70 “heavy” DNA [g/ml]

CsCl 1.72 “heavy” DNA

 Active degraders identified vie 1.74 T-RFLP fingerprinting of “heavy” (13C-labelled) DNA 12C-Toluene 13C-Toluene 1.76 Winderl, Penning, von Netzer, Meckenstock and Lueders. ISME J (2010) IdentificationIdentification ofof ClostridiaClostridia asas keykey toluenetoluene degradersdegraders Institute of Groundwater EInstitutcology

 Clostridia are typically known as fermenting and often also as pathogenic microbes  Here we show that members of the Clostridia also are of yet-unrecognised importance in BTEX degradation in aquifers

BACTERI

BETAPRO- LA07Ba35 LA07Ba46 “heavy” uncultured LA07Ba34 clostridial toluene degraders LA07Ba29 LA07Ba39 (146 & 178 bp T-RFs) LA07Ba11

e Desulfosporosinus sp. Y5, AY233860 a e Desulfosporosinus orientis, Y11571 c Desulfosporosinus sp. S10, AF076527 a c ‘Deferrispora aromatica’ UKTL, EU711071 c ) LA07Ba14 o a LA10Ba25 idi toc r acetoxidans, Y11566 p t niger, X55797

s CLOSTRIDA e o LA10Ba46 P l C Desulfotomaculum thermobenzoicum, L15628 ( Toluene-degrading consortium clone Eub1, AF423181 Desulfotomaculum sp. Ox39, AJ577273

Winderl, Penning, von Netzer, Meckenstock and Lueders. ISME J (2010)