MARBioFEED – Enhanced Biorefining Methods for the Production of Marine Biotoxins and Microalgae Fish Feed

The global demand for safe seafood increases due an expanding population and an increased awareness that fish and shellfish are a healthy source of food. With increased pressure on land-based agriculture due to climate change, the potential for sustainable, nutritious marine sourced food is growing. The quality assurance of fish and shellfish for human consumption is of paramount importance in ensuring the marketability of products from this expanding and predominantly rural sector. The industry and regulators rely heavily on various analytical tools to guarantee safety for human health. With more than 26 EU regulated algal toxins, there is a need for available analytical standards for these biotoxins. This project focuses on large-scale algal biotechnology with the goal of producing marine algal toxins for reference materials and safe algal supplement for fish feed.

The project has resulted in the development of enhanced methods for the purification of several important algal toxins from both microalgae and contaminated shellfish. These purified toxins will be used as calibrant standards for national monitoring programs and for other areas of research e.g., evaluation of toxicity, pharmacology, development of bioassays, etc. Significant progress has been made with respect to the development of optimized procedures for enhancing the growth and toxin production of the microalgae. In addition to the known algal toxins some new algal metabolites have been identified and attempts will be made to purify and fully characterize these new compounds. Methods have been developed for extraction of algal toxins from different matrices. This should improve the effectiveness of some of the shellfish testing procedures, and maybe useful for purification of toxins.

Fish feed supplemented with 20% of microalgal biomass (Nannochloropsis gaditana) was prepared by Neoalgae in Spain. This product is currently being marketed on their website. The feed is targeted toward juvenile fish to support survival, growth and immunity. A fish feeding trial was performed on juvenile trout, however no significant differences were observed in the survival, growth and fatty acid content of the fish fed the supplemented feed compared with the control. It was noted that the fish did not feed as well on the supplemented feed compared to the control.

MARBioFEED-team at IEO in 2016

MARBioFEED is a four year project (2016-2019) supported by the First Call for Transnational Research Projects within the Marine Biotechnology ERA-NET, involving partners from the Marine Institute in Ireland, the Norwegian Veterinary Institute in Norway, the Spanish Oceanographic Institute and Neoalgae in Spain and the National Research Council in Canada.

Project participants at Norwegian Veterinary Institute:
Ingunn Anita Samdal
Morten Sandvik
Christopher O. Miles
Kjersti Løvberg

Presentations and publications

To date the project has led to the publication of 11 peer reviewed papers and 21 conference presentations. A further one manuscript have been accepted to a peer reviewed journal and at least four are in preparation.

Articles

  1. Samdal IA, Løvberg KE, Kristoffersen AB, Briggs LR, Kilcoyne J, Forsyth CJ, Miles CO. A practical ELISA for azaspiracids in shellfish via development of a new plate coating antigen. J Agric Food Chem 2019, 67, 2369–2376.
  2. Kilcoyne, J.; McCoy, A.; Burrell, S.; Krock, B.; Tillmann, U. Effects of temperature, growth media, and photoperiod on growth and toxin production of Azadinium spinosum. Mar Drugs 2019, 17, 489.
  3. García-Portela, M.; Reguera, B.; Ribera d’Alcalà, M.; Rodríguez, F.; Montresor, M. Effects of small-scale turbulence in two species of Dinophysis. Harmful Algae 2019, 89, 101654.
  4. Kenton,N.T., Adu-Ampratwum, D., Okumu, A.A., McCarron, P., Kilcoyne, J., Rise, F., Wilkins, A.L., Miles, C.O., Forsyth, C.J. Stereochemical definition of the natural product (6R,10R,13R,14R,16R,17R,19S,20S,21R,24S,25S,28S,30S,32R,33R,34R,36S,37S,39R)-azaspiracid-3 via total synthesis and comparative analyses. Angew Chem 2018, 57, 810‒813.
  5. Kenton, N.T., Adu-Ampratwum, D., Okumu, A.A., Zhang, Z., Chen, Y., Nguyen, S., Xu, J., Ding, Y., McCarron, P., Kilcoyne, J., Rise, F., Wilkins, A.L., Miles, C.O., Forsyth, C.J. Total synthesis of (6R,10R,13R,14R,16R,17R,19S,20R,21R,24S,25S,28S,30S,32R,33R,34R,36S,37S,39R)-azaspiracid-3 reveals non-identity with the natural product. Angew Chem 2018, 57, 805‒809.
  6. Miles CO, Kilcoyne J, McCarron P, Giddings SD, Waaler T, Rundberget T, Samdal IA, Løvberg KE. Selective extraction and purification of azaspiracids from blue mussels (Mytilus edulis) using boric acid gel. J Agric Food Chem 2018, 66, 2962‒2969.
  7. Sibat, M., García-Portela, M., Hess, P. First identification of a C9-diol-ester of okadaic acid in Dinophysis acuta from the Galician Rías Baixas (NW Spain). Toxicon, 2018,153, 19‒22.
  8. García-Portela, M.,Riobó, P., Reguera, B., Garrido, J.L., Blanco,J., Rodríguez, F. Comparative ecophysiology of Dinophysis acuminata and D. acuta: effect of light intensity and quality on growth, toxin content and photosynthesis. J Phycol 2018, 54, 899–917.
  9. Hernández-Urcera,J., Rial, P., García-Portela, M., Lourés, P., Kilcoyne, J., Rodríguez,F., Fernández-Villamarín, A., and Reguera, B., Notes on the cultivation of Dinophysis species and their ciliate prey Mesodinium. Toxins, 2018, 10, 505.
  10. García-Portela, M.; Reguera, B.; Sibat, M.; Altenburger, A.; Rodríguez, F.; Hess, P. Metabolomic profiles of Dinophysis acuminata and Dinophysis acuta using non targeted high-resolution mass spectrometry: Effect of Nutritional Status and Prey. Mar Drugs 2018, No. 16, 143.
  11. Kilcoyne, J.; McCarron, P.; Twiner, M. J.; Rise, F.; Hess, P.; Wilkins, A. L.; Miles, C. O. Identification of 21,22-dehydroazaspiracids in mussels (Mytilus edulis) and in vitro toxicity of azaspiracid-26. J Nat Prod 2018, 81, 885–893.

Posters

  1. Samdal IA, VanNieuwenhove I, Løvberg KLE, Kilcoyne J, Wright EJ, Sandvik M, Uhlig S, Miles CO.”Immunoaffinity columns for clean-up and concentration of algal toxins” 18th International Conference on Harmful Algae, Nantes, France, 21–26 October 2018.
  2. Burrell, S.; Kilcoyne, J.; Tillman, U.; Miles, C. O. Stable isotopic labelling of AZA and OA-group toxins. International Conference on Harmful Algae, Nantes, France, 21–26 October, 2018.
  3. Hernández-Urcera, J.; Rial, P.; García-Portela, M.; Lourés, P.; Rodríguez, F.; Fernández-Villamarín, A.; Reguera, B. Optimization of laboratory cultures of two Dinophysis species. International Conference on Harmful Algae, Nantes, France, 21–26 October, 2018.
  4. Kilcoyne, J.; Burrell, S.; Salas, R.; Silke, J.; Delgado, F.; Albert, I.; Canga, M.; McCarron, P.; Rodríguez Hernández, F.; Reguera, B.; Samdal, I. A.; Sandvik, M.; Miles, C. O. MARBioFEED – enhanced biorefining methods for the production of marine biotoxins and microalgae fish feed. Irish Algal Researchers conference, NUIG, Ireland, 6th September, 2018.
  5. Hernández-Urcera, J.; Rial, P.; García-Portela, M.; Lourés, P.; Rodríguez, F.; Fernández-Villamarín, A.; Reguera, B. Optimization of laboratory cultures of two Dinophysis species. IV International Symposium on Marine Sciences (ISMS)-REDIBAL, Vigo, Spain, 20–22 June, 2018.
  6. Kilcoyne, J.; Burrell, S.; Salas, R.; Silke, J.; Delgado, F.; Albert, I.; Canga, M.; McCarron, P.; Rodríguez Hernández, F.; Reguera, B.; Samdal, I. A.; Sandvik, M.; Miles, C. O. MARBioFEED – enhanced biorefining methods for the production of marine biotoxins and microalgae fish feed. ERA-MBT final conference, Oslo, Norway, November 21st, 2017.
  7. Kilcoyne J, Burrell S, Salas R, Silke J, Delgado F, Albert I, Canga M, McCarron P,Hernandez FR, Reguera B, Samdal IA, Sandvik M, Miles CO. “MARBioFEED – enhanced biorefining methods for the production of marine biotoxins and microalgae fish feed.” The 17th International Conference on Harmful Algae (ICHA), Florianopolis, Santa Catarina, Brazil, 9. – 14. October 2016.

Oral presentations

  1. Kilcoyne, J.; Burrell, S.; Salas, R.; Silke, J.; Delgado, F.; Albert, I.; Canga, M.; McCarron, P.; Rodríguez Hernández, F.; Reguera, B.; Samdal, I. A.; Sandvik, M.; Miles, C. O. “MARBioFEED – enhanced biorefining methods for the production of marine biotoxins and microalgae fish feed.” Research symposium, Marine Institute, Galway, Ireland, October 24th, 2019.
  2. Kilcoyne, J.; Burrell, S.; Salas, R.; Silke, J.; Delgado, F.; Albert, I.; Canga, M.; McCarron, P.; Rodríguez Hernández, F.; Reguera, B.; Samdal, I. A.; Sandvik, M.; Miles, C. O. “MARBioFEED – enhanced biorefining methods for the production of marine biotoxins and microalgae fish feed.” Shellfish safety workshop, Athlone, Ireland, October 8th, 2019.
  3. Kilcoyne, J.; McCoy, A.; Burrell, S.; Krock, B.; Tillmann, U. “Effects of light, temperature, and nutrients on growth and toxin production of Azadinium spinosum”. SITOX – 2nd Meeting on Natural Toxins. University of Parma, Italy, 18–19 Sept, 2019.
  4. Miles CO, Beach DG, Kerrin E, McCarron P, Kilcoyne J, Giddings SD, Waaler T, Rundberget T, Samdal IA, Løvberg KLE. “Boronate techniques for clean-up and concentration of diol-containing algal toxins.” 18th International Conference on Harmful Algae, Nantes, France, 21–26 October 2018.
  5. Kilcoyne, J.; McCoy, A.; Burrell, S.; Gaiani, G.; Salas, R.; Tillmann, U. “Effects of light, temperature, and nutrients on growth and toxin production of Azadinium spinosum, Prorocentrumlima, and Pseudonitzchia australis.” International Conference on Harmful Algae, Nantes, France, 25th October, 2018.
  6. García-Portela, M.; Reguera, B.; Rivera d’Alcalà, M.; Rodríguez, F.; Montresor, M. “The turbulent life of Dinophysis.” International Conference on Harmful Algae, Nantes, France, 23. October, 2018.
  7. Miles CO. “Structures and chemistry of the azaspiracids, and their consequences for azaspiracid toxicity.” SITOX – First Meeting on Natural Toxins. Padova, Italy. 6. – 7. Sept 2018.
  8. Kilcoyne, J.; McCoy, A.; Burrell, S.; Gaiani, G.; Salas, R.; Tillmann, U. “Effects of Light, Temperature and Nutrients on Growth and Toxin Production of Azadinium spinosum, Prorocentrumlima, and Pseudonitzchia australis.” Irish Algal Researchers conference, NUIG, Ireland, 6th September, 2018.
  9. García-Portela, M.; Reguera, B.; Rivera d’Alcalà, M.; Rodríguez, F.; Montresor, M. “The turbulent life of Dinophysis.” IV International Symposium on Marine Sciences (ISMS)-REDIBAL, Vigo, Spain, 20–22 June 2018.
  10. Kilcoyne, J.; Burrell, S.; Salas, R.; Silke, J.; Delgado, F.; Albert, I.; Canga, M.; McCarron, P.; Rodríguez Hernández, F.; Reguera, B.; Samdal, I. A.; Sandvik, M.; Miles, C. O. “MARBioFEED – enhanced biorefining methods for the production of marine biotoxins and microalgae fish feed.” ERA-MBT final conference, Oslo, Norway, November 21st, 2017.
  11. Kilcoyne J, Burrell S, Salas R, Silke J, Canga M, Hernandez FR, Reguera B, Samdal IA,Sandvik M, Miles CO. “MARBioFEED – enhanced biorefining methods for the production of marine biotoxins and microalgae fish feed.” 11th International Conference on Molluscan Shellfish Safety (ICMSS). Galway, Ireland, 14.–18. May 2017.
  12. Samdal IA, Leonardo S, Campas M, Rambla M, Diogene J, Briggs LR, Forsyth CJ, KilcoyneJ, Miles CO,”Immunoassays for the detectionof the marine biotoxin azaspiracids.” 11th International Conference on Molluscan Shellfish Safety (ICMSS). Galway, Ireland, 14.–18. May 2017.
  13. Samdal IA, Sandvik M, Løvberg,KE, Briggs LR, Forsyth CJ, Xu J, Kilcoyne J, McCarron P, Miles CO. “Development of immunoaffinity columns for purification of the marine toxins azaspiracids.” 6th International Symposium of Marine and Freshwater Toxins Analysis. Baiona, Spain, 22.-25. October 2017.
  14. Kilcoyne, J.; Burrell, S.; Salas, R.; Silke, J.; Delgado, F.; Albert, I.; Canga, M.; McCarron, P.; Rodríguez Hernández, F.; Reguera, B.; Samdal, I. A.; Sandvik, M.; Miles, C. O. “MARBioFEED – enhanced biorefining methods for the production of marine biotoxins and microalgae fish feed.” ERA-MBT stakeholder meeting, Brussels, October 13th, 2016.