Exploring novel DNA-based autonomous platform for marine water monitoring

Transcription

1 A. Krolicka, K. Knapik, A. Bagi, M. Mæland, C. Boccadoro and T. Baussant - IRIS C. Preston, J. Birch and C. Scholin - MBARI S. Soelberg and C. Furlong Univ. Washington Exploring novel DNA-based autonomous platform for marine water monitoring Opportunities for petroleum and aquaculture industry

2 Environmental biosensors IRIS working with environmental issues, particularly related to biological effects and monitoring of e.g. petroleum activities Biological changes = Mean to address changes in ecosystem structure and function «Real time» biology = biosensors Environmental biosensors use of DNA use of living organisms (BiotaGuard) use of natural marine microbes as biosensors

3 Environmental Sample Processor fully automated platform for species detection based on near real-time gene-based assays «Lab in a can» Image: Holly Bowers 2014 MBARI 10. mars

4 Analytical modules on 2G-ESP current version for seawater Molecular probing analysis in situ (qpcr & SHA) SHA vizualization 10. mars

5 Microbes The ESP can detect a wide range of targets based on their gene signatures Harmful Algae Invertebrate Larvae Pseudo-nitzschia sp. (toxic & nontoxic) Heterosigma akashiwo (& other raphidophytes) Balanus glandula (Acorn barnacle) Osedax Mytilus sp. (Shore mussels) Toxins Alexandrium tamarense/ catenella Karenia sp. Haywood et al Journal of Phycology Jones et al Molecular Ecology Notes Mikulski et al Harmful Algae Preston et al Environmental Microbiology Carcinus maenus sp. (Green crab) Polychaete

6 Areas of potential interest for petroleum and aquaculture industry Biodiversity assessment in compliance with regulation Bioindicator species of «events» Search for rare, difficult to collect or invasive species Early-warning detection of toxic/diseasecausing species including algae, parasites and pathogens 6

7 INTSOK Murmansk 17 September Program Operational aspects in Northern areas Leak detection/moab project Petromaks MOAB Leak detection The primary objective of this project is to adapt an Environmental Sampling Processor (ESP) developed at MBARI to the real-time detection of an array of oil-related bacteria used as markers of hydrocarbon occurence in the water 7

8 Succession of Hydrocarbon-Degrading Bacteria following hydrocarbons release The deep ocean microbial community is highly sensitive to the presence of released hydrocarbons and is an effective biosensor for contamination in this environment. (Dubinsky et al., 2013; Environmental Science & Technology) Deepwater Horizon Oil Spill

9 Preliminary work: proof of concept Microbial 16S rrna Q-PCR (Krolicka et al., 2014) AMOP, Canada 2014

10 Which species are there? Krolicka et al. (in prep) 10

11 Work flow for microbial detection related to oil in water with ESP 11

12 Bench assay Mesocosm oil leakage ESP robotized assay Seawater collection 12

13 abundance per ml seawater Oil Concentration mg/l 1,0E+07 ESP qpcr Results 6 1,0E ,0E ,0E+04 1,0E+03 1,0E Olei16S - O Olei16S - C Colwell16S - O Colwell16S - C CyclogryB - O IPC (Ct) Oil Concentration 1,0E ,0E Time 0

14 ESP versus Traditional Lab Methods in the Detection of Microbes in Oil-Contaminated Seawater Illustration: Chris Preston (MBARI) All groups detected with traditional lab methods were detected on the ESP. Oleispira- good match between ESP & Bench Others- ESP underestimates, but most within an order of magnitude Biggest issue with overcoming qpcr inhibition caused by the sample matrix

15 Detecting the microbial response to addition of oil to native seawater on ESP Arrays Target Probes Bacterioplankton Array Control Probes Illustration: Chris Preston (MBARI) A general bacterioplankton array showed an increase in microbial rrna belonging to specific groups in oil enriched mesocosms. Under development: Specific SHA probes for Oleispira, Colwellia, DWH Oceanospirales which were shown to respond within 48hrs after addition of oil. Use of other probes e.g. SAR11 clade

16 Existing 2G ESP ESP Development Trajectory 3G ESP under development now Focus on limited # of targets with broad taxonomic affiliation First application of qpcr below the ocean surface (to 1600m+) Ecogenomic sensor concept validation Enable mobile operations Expand repertoire of analytical modules (SPR and ddpcr) Focus on larger # of targets

17 RCN GENOMAPE (Petromaks 2) 17

18 RCN ISMOTOOL (Havbruk) Building on experience from MOAB and GENOMAPE Specific DNA-based assays will be developed in the laboratory using protocols mimicking ESP for present environmental challenges for the aquaculture industry (1) fish pathogen (amoebic gill disease AGD) and parasite (salmon lice) in the water column (2) escaped farmed fish (edna of Atlantic salmon and rainbow trout) Field demonstration with ESP IRIS Laboratory work and q-pcr bench assays NVI DTU 18

19 Advantages of ESP Design for water column samples Sample collection, preparation, analysis and preservation are fully autonomous Access in real-time with typically ~2-3h lag time qpcr (amplification, for low abundant species) and SHA (no amplification; for more abundant species) Archiving also samples for post-retrieval analysis 19

20 Challenges with current ESP Relatively complex operation Needs expertise Cost q-pcr inhibition ( droplet digital PCR) Sample volume Typically 2 L can be challenging for rare species

21 Conclusion ESP is a unique, mature, off the shelf technology for water analysis with several applications for research, regulation and industry needs Development from 2G- to 3G-ESP will open up for new opportunities and capabilities to monitor the ocean for ecosystem management Image souce: Kelly Lance (c) 2014 MBARI NDP workshop NOG, Oslo 21

22 Thank you NDP workshop NOG, Oslo 22