Christoff er Bugge Harder, Lea Ellegaard, Berith E. Knudsen, Søren Rosendahl, Flemming Ekelund, Christian Albers and Jens Aamand

Terrestrial Ecology, University of Copenhagen

GEUS (Geological survey of Denmark and Greenland)

MIRESOWA

(MIcrobial REmediation of SOil and WAter

The artificial lake bottoms on water treatment plants

- analysed by 454/pyrosequencing

Overall interest of MIRESOWA is bioremediation: removal of pesticides by microorganisms

Bioaugmentation: Cleaning up contaminated sites by addition of a pesticide mineralising bacterial strain

Often difficult to get an extraneous strain to survive and flourish in a natural environment (with competing strains and predatory larger microorganisms)

Denmark obtains almost all its potable water from minimally treated groundwater (big national pride!)

Background about MIRESOWA

I work mostly on free-living protozoa

- quantitatively important bacterivores

- Predation on pesticide degrading bacterial strains

- may either increase or decrease the bacterial activity

- Higher trophic level interactions?

Water treatment plant sand filters

Water treatment plant sand filters

- constant temperature, high oxygen- frequent backflushing (in/outflow)- darkness (little if any photosynthesis)- primary production by nitrifying and Mn/Fe oxidising

bacteria- Artificially , stable and controllable - In Denmark, some have problems with pesticides

(POPs)• Main research questions:• Are filters useful as bioaugmediation sites?

• Are they a predominantly (preferably) sterile artificial environment with the occasional pathogenic invader that must be exterminated?

or• A seminatural ecosystem whose diversity we will have to

deal with (or manipulate intelligently)?

Nitrobacter sp.

Galionella ferruginea

11 Danish plants sampled

Landet (Svendborg)Lunde (Svendborg)Odense H (VC syd)Lindvedværket  (VC syd)Aike (Esbjerg)Astrup (Esbjerg)Islevbro (KE)Søndersø (KE)Vejle S (Tre-For)Svenstrup (Tre-For)Hvidovre (KE)

From top layer + 20-80cm below + pre/postfilter (if possible) , 22 samples total

Sampling from waterworks

- Closed and open filters- Some with both pre- and post-filter (2 steps)- Drilling depths 10-175 meter- With and without pesticide contaminations

“Sand” filter materialMost often coal or

quartz Very different in size

and shape (0,5 to 10 mm in diameter)

With or without coating (most commonly Fe 3+ and/or manganese)

PCR of 18S

Baldauf 18S phylogeny. (PNAS 2008)

All 18S “eukaryote specific” primers have biases

- An ecological group

- Difficult to identify (DNA often needed)

DNA extraction, PCR + cloning + sequencing

We designed another general eukaryote primer set of 500-550 bps (covering V1+V2+V3 hypervariable regions)

Slightly better for protists than e.g SSU_Fo4+SSU_R22

126 of 135 clones verified 18S eukaryotes by BLAST

DNA extraction, PCR and pyrosequencing

• DNA extracted in quadruplicates and pooled for PCR

• Libraries constructed by 2-step PCR, analysed in MOTHUR (Schloss

et al. 2009)

•

• 264080 +400 bp reads (477bp median, singletons discarded, no

ambiguos bases, maxhomop=8, q>25, checked with uchime)

• >90% of those were target 18S

Processing of the18S 454 run “Unlikely” organisms (spotted hyena, Homo, Lolium + other

higher plants) removed before analysis (12.3% of reads) Resampling necessary – to 160 reads to capture all 22 plants,

to 4541 to capture 13 plants to (near)-saturation

No major qualitative differences between the two resamplings

1125 OTUs (3% level) across 13 saturated plants Many sequences difficult to assign confidently (47%

unassigned by Silva (it´s a poor database, I know!))

Most numerous OTU?Freshwater sponges?5 OTUs (at 3%),

about 20% of all sequences

(NB!! but only 82-85% similarity to top BLAST hits!)

Absent from 2 plants

Many protozoa found are unequivocal at genus level (+95-99% BLAST similarity)

Rhogostoma sp.

Naegleria sp.

Centropyxis laevigata

Lacrymaria sp.

Lecythium

Acineta sp.

Other eukaryotes

Debaryomyces sp.

Athalamea sp.

Chaetonotus sp.

Nematods (Monhysterids)

Gammarus sp.Synchrytrium sp.

Acaulopage sp.

Rotifera sp.

Characteristics of the eukaryotic communitiesOTUs dominated by protozoa

All eukaryotic supergroups foundSome potentially pathogenic amoeba

(Hartmannella, Vexilifera, Naegleria) – but common everywhere in soil and water

Characteristics of the eukaryotic communities

NMS on 22*160 reads

No obvious geographical structure

Large local variations/ differences (surface/ subsurface layers, pre/ post-filters)

OTU diversity appear to be most important factor

Food chain (trophic levels)

Bacteria.

Food chain (trophic levels)

Bacteria.

Cercomonas sp.(flagellate)

Food chain (trophic levels)

Bacteria.

CiliatesCercomonas sp.(flagellate)

Food chain (trophic levels)

Bacteria.

Ciliates

Chaetonotus sp.

(Nematodes?)

Cercomonas sp.(flagellate)

Food chain (trophic levels)

Bacteria.

Ciliates

Chaetonotus sp.

(Nematodes?)

Cercomonas sp.(flagellate)

Last trophic level absent from several treatment plants

Food chain (trophic levels)

Bacteria.

CiliatesChaetonotus sp.

(Nematodes?)Potential implications for bioaugmentation in water treatment plants:

- Positive isolated effect on bacterial naphtalene mineralisation of ciliate grazing (Tso & Taghon 2006)….- ….but adding further trophic levels gave an adverse effect on mineralisation (Näslund et al. 2010)

Cercomonas sp.(flagellate)

Questions and to-do-listTry a better taxonomic annotator (Jaguc –

suggestions welcome)Fauna primarily from groundwater or air

dispersed?More detailed b-diversity comparisons

between plantsDatasets for comparisons with e.g. the net

related index (NRI) – suggestions/ideas utmost welcome

Preliminary conclusionsSand seminatural ecosystem whose

diversity we will have to deal with (or manipulate intelligently)

Trophical interaction system resembling lake/soil sediments (with lower diversity)

Danish water treatment plants are likely very well suited for field scale bioremediation experiments- but some are better than others, - andit will be necessary to find strains that can

tolerate recurring flush-outs……

Acknowledgements Flemming Ekelund, Terrestrial Ecology, University of Copenhagen Søren Rosendahl, Terrestrial Ecology, University of Copenhagen Lars H. Hansen, Microbiology, University of Copenhagen Karin Vestberg, Microbiology, University of Copenhagen Berith Knudsen, GEUS (Geological survey of Denmark and

Greenland) Jens Aamand, GEUS Lea Ellegaard, GEUS Christian Albers, GEUS Tre-For Vand,VandCenter Syd, Københavns Energi, Esbjerg

Forsyning, Svendborg Vand (Water companies) Danish Strategic Research Council

……and of course, the BSPB – and thank you for the attention5!

OTU separation value importance

• Using 10% does change the overall picture presented here