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Christoffer Bugge Harder, Lea Ellegaard, Berith E. Knudsen, Søren Rosendahl, Flemming Ekelund, Christian Albers and Jens Aamand Terrestrial Ecology, University.

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Presentation on theme: "Christoffer Bugge Harder, Lea Ellegaard, Berith E. Knudsen, Søren Rosendahl, Flemming Ekelund, Christian Albers and Jens Aamand Terrestrial Ecology, University."— Presentation transcript:

1 Christoffer 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

2 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!)

3 -quantitatively important bacterivores -Predation on pesticide degrading bacterial strains -may either increase or decrease the bacterial activity -Higher trophic level interactions?

4

5 -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

6            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

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

8 Most 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)

9 Baldauf 18S phylogeny. (PNAS 2008) All 18S “eukaryote specific” primers have biases -An ecological group -Difficult to identify (DNA often needed)

10 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

11 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

12 “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!))

13 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

14 Rhogostoma sp. Naegleria sp. Centropyxis laevigata Lacrymaria sp. Lecythium Acineta sp.

15 Debaryomyces sp. Athalamea sp. Chaetonotus sp. Nematods (Monhysterids) Gammarus sp. Synchrytrium sp. Acaulopage sp. Rotifera sp.

16 OTUs dominated by protozoa All eukaryotic supergroups found Some potentially pathogenic amoeba (Hartmannella, Vexilifera, Naegleria) – but common everywhere in soil and water

17 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

18 Bacteria.

19 Cercomonas sp. (flagellate)

20 Bacteria. Ciliates Cercomonas sp. (flagellate)

21 Bacteria. Ciliates Chaetonotus sp. (Nematodes?) Cercomonas sp. (flagellate)

22 Bacteria. Ciliates Chaetonotus sp. (Nematodes?) Cercomonas sp. (flagellate) Last trophic level absent from several treatment plants

23 Bacteria. Ciliates Chaetonotus 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)

24 Try a better taxonomic annotator (Jaguc – suggestions welcome) Fauna primarily from groundwater or air dispersed? More detailed  -diversity comparisons between plants Datasets for comparisons with e.g. the net related index (NRI) – suggestions/ideas utmost welcome

25 Sand 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……

26 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!

27 Using 10% does change the overall picture presented here


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