Projects:Dépollution des stands de tirs Suisses

Dépollution des sols des stands de tir Suisses

Des centaines de tonnes de métaux lourds contaminent les abords des stands de tir suisses. Leur assainissement devrait couter près de 900 millions de francs aux autorités (référence).

Mining nature

Mining nature is about the identification of new open access tools for pollution detection. The logic behind the project is this: we hope (as it should) that the selective pressure applied to an environment by the presence of a toxic compound led to evolve a set of specific molecular tools allowing the local microbial community to overcome this stress. Bacteria capable to grow in presence of high concentration of pollutant will most likely, have some of those molecular tools. Clear until now is the resemblance with the antibiotics, molecules that fungi produce to get rid of competitors (competition stress), and with the antibiotic resistance, mechanisms that the competitors uses to fight back the fungi. There are two ways to induce resistance: extrude the stress or from the cellular environment or metabolize it to a safe form. While molecules like antibiotics (but also other type of compounds like benzene derivatives) can be degraded in smaller molecules heavy metals atoms (lead, mercury and etc) cannot (unless using a nuclear reactor!). The two strategies in this case are as we said, extrusion or bio-accumulation. The latter being the undetermined storage of the pollutant in the cell complexed as a non toxic form. If you choose this way then you have to remove the bacteria from the soil/water/whatever that is normally a quite difficult task . Much better is thus to use plants. In the future having a bacterial based detector system would allow screening for the best bio-accumulator plants. Once again the problem is moved forward from the soil to the plants, with the difference that a already concentrated quite a lot the pollutant, a bacterial digestor and subsequent further concentration may be applicable.

Who's on board

Benoît Dubuis : CEO du Campus Biotech Genève Carmelo Bisognano : general manager d'INARTIS Davide Merulla : spécialiste de la détection de polluants basée sur des bioreporteurs cellulaires Luc Henry : président - co-fondateur d'Hackuarium Yann Heurtaux : community builder d'Hackuarium Clément Épié : ecosytem catalyst pour La Paillasse Nicolas Loubet : ecosytem catalyst pour La Paillasse Clément Drevo : étudiant, Genève, travaillera à plein temps sur le projet en temps que stagiaire de janvier 2014 jusqu'à fin avril 2014 (4 mois) Charlotte Rizzo (Paillasse Saône / politiques publiques innovantes & gestion de déchets) : possibilité de mobiliser des énergies en région Rhône Alpes (exploration sur place ou aide à la recherche), et de participer au projet à la mesure des compétences requises Sachiko Hirosue : the team at biodesign for the real world (currently focused on arsenic reporter, working with Davide's PhD mentor) Anne-Laure Pittet : biologiste

À contacter : Sénamé Koffi (WoeLab / grosse expertise sur les pratiques l'upcycling à partir de déchets). Todd Huffman (qui a travaillé avec un hacker en Afghanistan qui s'est penché sur le sujet) A Lyon : Emiel van der Kouwe (biologiste, pharmacien de la paillasse saone), Agnès (labos bio de synthèses à l'INSA en charge de projets de dépollutions), Carole Barbier (chimiste déchets), Marion Picard (CERN), Frédéric Rees (doctorant en dépollution des sols par charbon actif stocké) Karl Perron : BioOutils - UNIGE

Key questions

QUESTIONS

1. How high is (too) high ? there is no real answer we have to make replica plates with logarithmic increasing concentration of pollutant to understand that. Basically, we need a lot of plates.

2. How do I identify which type of bacteria it is? The best would be to sequence the 16s tRNA region, a commonly used sequence conserved amongst most of the microorganisms with only minor but specific mutation. People doing that are using degenerated primers specific for those areas so that the PCR and subsequent sequencing can be performed even without knowing the exact sequence already . It is tricky but there is a lot a literature about it -> Let's try to focus on low-cost methods first. PCR only based methods? Or collaboration with genomic institutes for outsourcing the sequencing part.

Having easy access to a collaboration with a genomic facility may be nice but really not fundamental. The information we can get with some 16s sequencing followed by a comparison with the information within public databases should be more than enough for us.

3. Can we directly use those bacteria to measure the presence of a pollutant? As Carmelo was saying we can already use this bacteria as indicator. The growth rate of bacteria slows down with the increasing amount of stressor. It is possible to measure the growth speed of a culture using turbidity as indicator and produce a so-called growth curve. By interpolation, comparing the growth curve of the sample with a calibration curve it is possible to deduce the initial concentration of pollutant. We need a turbidometer and a lot of tubes, and off course the resistant bacteria.

CONSIDERATIONS ABOUT THE MOLECULAR HANDLING : GMO

Having the resistant bacteria means having in it a collection of molecular tools specific for the compound or for a class of similar molecules. The most accessible application is to produce a biosensor connecting the sensing element to the production of a so-called reporter gene (what I do btw :) ).

WISH LIST & CONCERNS

-we need tons of plates and media

-we need the pure toxic compound

-we need a -80°C or a lyophilizer (a spedvac is not what we need)

-there may be a problem concerning the waste we are going to produce if we screen for heavy metals (needs to be sorted out)

-the PCR screening will be need when we're going to detect some resistant strain to confirm if we have found something new (which is much better :) )

RESOURCES AND STRATEGY

- A good starting point may be a map of the polluted sites so that schools, associations and private can pick their preferred one.

- Second we have to set up the method and Clément Drevo could definitely be a huge help here: if we want to involve schools we need to have a slightly functional working package while clearly leave room and support for further development.

Deployment

1.Les tout premiers pas

• Visite d'un stand de tir / recueil d'échantillons / analyse dans le lab d'Hackuarium

Crowdsourcing? (classes, citoyens) ou expéditions (membres de la communauté)?

• Envoyer quelqu'un pour pitcher le projet au Forum ARPEA (L'Education à l'environnement)(pour toucher des amateurs qui veulent s'impliquer sur un projet d'exploration de ce type) => ABANDONNE
• Faire une liste précise des achats liés à ce projet (boite à petri, milieu,..)
• Mettre en place un endroit stérile et faire des tests de contamination de Hackuarium à vide (pour éviter les faux postifs au moment des tests) / 2014.08.27
• 1h d'exposition à 5 endroits du labo (plus un échantillion test) Test de contamination: YPD medium petri

2.Un peu de bibliographie

Soil sampling video - DIY soil sampling corer https://www.youtube.com/watch?v=1FG9AwTGr0o

Lead (Pb2+ and Pb4+) in shooting ranges

• Literature search about universal media for ground bacteria

http://www.sigmaaldrich.com/analytical-chromatography/microbiology/microbiology-products.html?TablePage=17996747

• General Medias that seem interesting / not complicated to make DIY

Nutrient Agar No 2, Vegitone (500g 141.00CHF)

[ http://www.sigmaaldrich.com/catalog/product/fluka/70148?lang=fr&region=CH Nutrient Agar] (100g 46.70CHF, 500g 200.50CHF)

• Home made

-Agar Agar (Migros) = 78 CHF per kilo

-Cenovis (Migros) = 40 CHF per kilo

-Bouillons de viande (Migros) = 26 CHF per kilo

-480 plates = 40 CHF

• for 1L (40 plates):

-16g bouillon = 0.4 CHF

-2g of Cenovis = 0.08 CHF

-15g agar = 1.20 CHF

-40 plates = 3.30 CHF

-total = 5 CHF per 40 plates => 0.13 CHF per plate

COSTS:

-480 plates = 12 L

-180g of agar agar = 15 CHF

-200g of bouillon = 5 CHF

-24 g of Cenovis = 10 CHF

-480 plates = 40 CHF

-Total = 70 CHF

• Literature about lead resistant bacteria:

-Kafilzadeh et al 2012 Euro. J. Exp. Bio. 2, 62-69 (gaz station sampling, Iran)

-Gummersheimer and Giblin 2003 Bios 74, 48-54 (lead factory sampling, USA)

-REVIEW: Naik and Dubey 2013 Ecotox Env Safety 98, 1-7

• Nutrient Agar No 2, Vegitone

composition

-agar, 15.0 g/L

-peptone (vegetable), 5.0 g/L

-vegetable extract, 3.0 g/L

-final pH:7.0±0.2 (25 °C)

• composition

-agar, 15 g/L

-meat extract, 1 g/L

-peptone, 5 g/L

-sodium chloride, 5 g/L

-yeast extract, 2 g/L

-final pH 7.1±0.2 (25 °C)

Effects of Growth Medium, Inoculum Size, and Incubation Time on Culturability and Isolation of Soil Bacteria > The media here seems to be very defined and complicated to find all components

• DIY media approaches - BioStrike project has started some: http://tinyurl.com/q2x6bol

-get the ingredients

-agar = agar agar

-meat extract = bouillon Kubor

-peptone = hydrolyzed whey protein? http://www.bodybuilding.com/fun/which-protein-will-help-you.html

-yeast extract = cenovis (CH) marmite (http://en.wikipedia.org/wiki/Marmite)

• for 250ml

-agar: 3.8(g)

-knorr beef bouillon: 4.3(g)

-cenovis: 0.5(g)

-peptone: 1.3(g)

• 3 weeks later, this is found to be contaminated -

-http://teach.genetics.utah.edu/content/gsl/html/recipes.html

-get the pure compound that we want to detect in a form that is soluble and bioavailable

3- Dispositifs d'innovation

• Petit kit pour diagnostiquer le niveau de pollution (inspiration)
• Kit pour construire son drône aérien (voir) => Hakim (FlyLab)
• Rassembler/construire le matériel nécessaire décrit par Davide

-OpenPCR

-GoGoFuge

-Dremelfuge

-Fluorimètre

4- Effort de mise à l'échelle

• Carte de tous les stands de tirs en Suisse
• Voir avec Ubimix / E. Haguet
• OpenData.ch for a gov source? Voir.
• Make.OpenData.ch project: swiss_army_contaminated_sites
• Use OpenStreetMap to locate? OSM.ch
• Stand de tir région vaud: PDF
• Oleg suggests the following 'In terms of tools, my philosophy is: use the absolute simple thing that works. For example right clicking on http://map.geo.admin.ch/ and pasting the resulting WGS84 coordinates into a shared Google Spreadsheet should work like a charm.'
• Carte des lieux pollués au métaux lourds en Suisse
• For Arsenic in Switzerland:
• Cartographie de la pollution des sols dans le monde
• Ground water movements are important: EAWAG
• Site:

-Crissier

-bioreporter Antimony and Arsenic as GMO

-bioremediation - bioabsorption /

20141013 Notes from meeting

• Based on Davide's calculation, all included, it would cost 0.18 CHF per plate.
• sampling -> keep cold (O/N ok, prevent drying) -> homogeneisation -> plating
• plating (triplicated) on three medium dilution (1x, 0.1x, 0.01x), incubate at 25-30°C + control
• -> choose medium according to growth and cost
• use soluble salts of heavy metals (polluants)
• Lead disposal / plates (at about 0.5g/kg)
• Before Friday:

-mixer (Luc, Gloryland)

-scale (Luc, Gloryland)

-toothpicks

-soap for dishes

-sponges

-alu foil

-agar agar 180g

-bouillon 200g

• Friday

-sterilize

-media

-toothpicks

• for Saturday -

-plates (Davide)

-alcohol 70% (SH)

-spatula (Davide)

-autoclave tape (have)

-markers (have)

-kitchen paper (have)