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
 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!)
- quantitatively
important
bacterivores
- Predation on
pesticide degrading
bacterial strains
- may either increase or
decrease the bacterial
activity
- Higher trophic level
interactions?
-
constant temperature, high oxygen
frequent backflushing (in/outflow)
Nitrobacter sp.
Galionella
ferruginea
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)?
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
-
Closed and open filters
Some with both pre- and post-filter (2 steps)
Drilling depths 10-175 meter
With and without pesticide contaminations
 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)
- An ecological
group
- Difficult to
identify (DNA
often needed)
Baldauf 18S
phylogeny.
(PNAS 2008)
All 18S “eukaryote specific” primers have biases
 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
 “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!))
 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
Naegleria sp.
Acineta sp.
Rhogostoma sp.
Lacrymaria sp.
Centropyxis
laevigata
Lecythium
Chaetonotus sp.
Athalamea sp.
Debaryomyces sp.
Acaulopage sp.
Rotifera sp.
Nematods
(Monhysterids)
Synchrytrium sp.
Gammarus sp.
 OTUs dominated by protozoa
 All eukaryotic supergroups found
 Some potentially pathogenic amoeba (Hartmannella,
Vexilifera, Naegleria) – but common everywhere in soil
and water
Characteristics of the eukaryotic
communities
 No obvious
geographical structure
 Large local variations/
differences (surface/
subsurface layers, pre/
post-filters)
 OTU diversity appear
to be most important
factor
NMS on 22*160 reads
Bacteria.
Cercomonas sp.
(flagellate)
Bacteria.
Cercomonas sp.
(flagellate)
Bacteria.
Ciliates
Cercomonas sp.
(flagellate)
Chaetonotus sp.
Ciliates
(Nematodes?)
Bacteria.
Cercomonas sp.
(flagellate)
Chaetonotus sp.
Ciliates
(Nematodes?)
Last trophic level absent from
several treatment plants
Bacteria.
Cercomonas sp.
(flagellate)
Bacteria.
Chaetonotus sp.
Ciliates
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)
(Nematodes?)
 Try a better taxonomic annotator (Jaguc –
suggestions welcome)
 Fauna primarily from groundwater or air dispersed?
 More detailed b-diversity comparisons between
plants
 Datasets for comparisons with e.g. the net related
index (NRI) – suggestions/ideas utmost welcome
 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……
 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!
• Using 10% does change the overall picture presented here