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DIATOMS AS FEED FOR AQUACULTURE




        By John J. Perez C.
Centric diatom                  Pseudo-nitzschia
Thalassiosira

                                                      Pennate diatom

                                 siliceous hoops (girdle bands)




Pores are species specific

                                  Greek diatomos, ‘cut
                                        in half ’

                             chlorophyll a, fluorescing red when
                             illuminated with blue Light
Diatoms support the most
   productive fisheries
Crucial role of diatoms in the global carbon cycle
Thalassiosira pseudonana (34Mb)
Phaeodactylum tricornutum
        (27 Mb)
Fragilariopsis cylindrus
                                                (80 Mb)




Fragilariopsis cylindrus (Grunow in Cleve &
Möller) W. Krieger in Helmcke & Krieger 1954
Pseudo-nitzschia multiseries
          (300 Mb)
Unique combination of genes and metabolic pathways
                      distinguish diatoms




      Thalassiosira                                                  Two of the novel type of spores
                                                                     of Chaetoceros furcillatus
     nordenskioeldii




                             Enormous amounts of                     Thalassiosira taelata
Young auxospore of Pseudo-
nitzschia multiseries        diversity encapsulated
                                 within diatoms

              http://www.aquatic.uoguelph.ca/Human/Research/ijkl/kaczmarskai.htm
Life in the ocean waves
  Eukaryotic
phytoplankton                        Cyanobacteria




                                          Heterotrophic
  Archaea                                      bacteria




      viruses

                                            Protists
Benthic diatoms are the main food
  source for abalone post-larvae
monospecific cultures of benthic diatoms have successfully been
   employed as feed for the culture of abalone post-larvae
in Taiwan, abalone post-larvae grown in hatcheries are usually fed with natural
populations of benthic diatoms, following the traditional method of using biofilms of
   mixed benthic diatoms as the settlement substrata for abalone post-larvae in
                                     hatcheries
a photosynthetically active biofilm of diatoms, e.g. Gyrosigma spp

                                                            Biofilm of mixed centric diatoms
postlarvae of various abalone species have a better growth
 rate when fed with cultured diatoms than those fed with
           natural unknown species of diatoms




                                      Post Larval 'spat': After 2-3 weeks
                                      swimming around in the water column, the pelagic
                                      veliger larva loses its velium and metamorphoses into
                                      the post-larval stage (spat). It loses its positive
                                      phototaxis (movement towards the sunlight), moves
                                      down from the water surface, and settles on hard
                                      surfaces in intertidal and subtidal areas. Settlement is
                                      triggered by a chemical that is produced by Coralline
                                      algae and adult abalone. Abalone feed on Coralline
                                      algae and the presence of adults indicates a good
                                      habitat to live! The post-larva sheds its swimming
                                      hairs (cilia) and begins to develop the adult shell form.
                                      This is the first feeding stage where they start to feed
                                      on diatoms.
Carbajal-Miranda
               et al. (2005)
                                      Navicula
                                      incerta


Amphiprora
paludosa


                          Haliotis
                          rufescens
Daume et al. (2000)
                                     Cylindrotheca
                                     closterium




                    Haliotis rubra     Amphora sp.




Navicula sp., N. jeffr               Cocconeis sp
Gordon et al. (2006)
Haliotis discus




Amphora luciae


                    Navicula cf. lenzii
Haliotis iris
Achnanthes longipes



                      Kawamura et al. (1998)

Nitzschia ovalis


                                      Nitzschia sp
Cocconeis p.           Navicula britannica
Gallardo and
   Buen (2003)
  Mixed diatoms vs.
monospecific cultures of
      Navicula
  Haliotis asinina
little information
    concerning
 diatoms as feed
    for Haliotis
    diversicolor
Kawamura et al., 1998
 Poor and unpredictable performance
        of abalone postlarvae
differences in                 lipid,
the species of                 protein and
diatoms they                   extracellular
have been fed                  polymeric
                               substances
                               (EPSs)
Hypothetical cellular pathways and processes in iron-limited pennate diatom cells. All roman green or red type depicts
gene transcripts found to be up- or down-regulated, respectively. Italicized and underlined green type indicates
metabolites found to be enriched relative to total protein in iron-limited cells. DF, diffusion factor; EPS, extracellular
polymeric substances; FR, ferric reductase; HMA, heavy metal-associated; PCD, programmed cell death; ROS, reactive
oxygen species.
AIMS
• to improve the growth and the survival of
  abalone post-larval stages in a specific growth
  system using specific diatoms species
• a better understanding of their basic dietary
  requirements
  farmers attempting to produce mollusks and
       crustaceans from the larval stage
• high cost of producing live food (microalgae)
• up to 30% of the total cost of production
  (Valenzuela-Espinoza et al., 1999).
Alternative
• use immobilized
  microalgae
• can be cheaper than
  algae produced by
  traditional methods
• ready for use (Chen,
  2003).
Specific aims
• Survey the diversity of diatoms species found
  in H. diversicolor hatcheries.
• Isolate and develop a monoculture that could
  be immobilized and encapsulated in alginate
  beads for long-term storage as algal stock.
monospecific algal stocks used as feed for
cultivating the post-larvae of H. diversicolor
• Lipids
• Proteins
• EPS
  – soluble EPS
  – bound EPS
  – internal carbohydrate
  – residual carbohydrate
Collection and preparatory cultures of benthic diatoms


   Diatoms from small abalone
        hatchery ponds

    scraped from abalone post-larvae
  settlement substrates (plastic plates)


 collected in l L collection bottles
                                           Yean-Chang Chen, Ph.D.
                                             Associate Professor,
  placed in a cool icebox                      Department of
                                                Aquaculture,
                                           National Taiwan Ocean
                                                 University,
transported to the Algal Laboratory           Keelung, Taiwan
                                           Email: ycchen@mail.ntou.edu.
   of the National Taiwan Ocean                          tw
                                             b0232@ind.ntou.edu.tw
    University, Keelung, Taiwan            Tel: 886 2 24622192 ext. 5221
Martek 120-liter   Deep aerated tank used for the
                   culture of Nannochloropsis for
photobioreactor    aquaculture
Eicosapentaenoic acid 20:5(n-3)
(EPA, 5,8,11,14,17-cis-eicosapentaenoic acid)
                       EPA is an omega-3 fatty acid

• an n-3 C20-polyunsaturated fatty acid that is
  metabolically active. with a doublefrom the endstartingcarbonthe third
                        carbon atom
                                      bond (C=C)
                                                  of the
                                                          after
                                                                chain.




a carboxylic acid with a 20-carbon chain and five cis double bonds
the first double bond is located at the third carbon from the omega end.
EPA and its derivatives have proved beneficial in prevention
       and treatment of certain medical conditions

  • coronary heart
    disease
  • blood platelet                    EPA is effective
    aggregation                       also in arresting
                                      and minimizing
  • abnormal cholesterol              tumor growth
    levels
  • several carcinomas
EPA is currently sourced from fish oil
                   • No other sources are
                     commercially available.
                      – fluctuates in price and quality.
                      – contamination of fish oil with
                        pesticides and heavy metals.
                      – an alternative economic and
                        consistent source of EPA is
                        needed.
                      – Microalgae are one
                        potential source.
Annual EPA demand
demand is expected to increase                       dwindling
                                                     supplies of
                                                       fish oil




                                                     125
                                                     tones in
                                                     Japan
                 Current market price of EPA ethyl
                 ester (95% pure) about $650/kg

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Upou 350 aquaculture and lipids

  • 1. DIATOMS AS FEED FOR AQUACULTURE By John J. Perez C.
  • 2. Centric diatom Pseudo-nitzschia Thalassiosira Pennate diatom siliceous hoops (girdle bands) Pores are species specific Greek diatomos, ‘cut in half ’ chlorophyll a, fluorescing red when illuminated with blue Light
  • 3. Diatoms support the most productive fisheries
  • 4. Crucial role of diatoms in the global carbon cycle
  • 7. Fragilariopsis cylindrus (80 Mb) Fragilariopsis cylindrus (Grunow in Cleve & Möller) W. Krieger in Helmcke & Krieger 1954
  • 9. Unique combination of genes and metabolic pathways distinguish diatoms Thalassiosira Two of the novel type of spores of Chaetoceros furcillatus nordenskioeldii Enormous amounts of Thalassiosira taelata Young auxospore of Pseudo- nitzschia multiseries diversity encapsulated within diatoms http://www.aquatic.uoguelph.ca/Human/Research/ijkl/kaczmarskai.htm
  • 10. Life in the ocean waves Eukaryotic phytoplankton Cyanobacteria Heterotrophic Archaea bacteria viruses Protists
  • 11. Benthic diatoms are the main food source for abalone post-larvae
  • 12. monospecific cultures of benthic diatoms have successfully been employed as feed for the culture of abalone post-larvae
  • 13. in Taiwan, abalone post-larvae grown in hatcheries are usually fed with natural populations of benthic diatoms, following the traditional method of using biofilms of mixed benthic diatoms as the settlement substrata for abalone post-larvae in hatcheries a photosynthetically active biofilm of diatoms, e.g. Gyrosigma spp Biofilm of mixed centric diatoms
  • 14. postlarvae of various abalone species have a better growth rate when fed with cultured diatoms than those fed with natural unknown species of diatoms Post Larval 'spat': After 2-3 weeks swimming around in the water column, the pelagic veliger larva loses its velium and metamorphoses into the post-larval stage (spat). It loses its positive phototaxis (movement towards the sunlight), moves down from the water surface, and settles on hard surfaces in intertidal and subtidal areas. Settlement is triggered by a chemical that is produced by Coralline algae and adult abalone. Abalone feed on Coralline algae and the presence of adults indicates a good habitat to live! The post-larva sheds its swimming hairs (cilia) and begins to develop the adult shell form. This is the first feeding stage where they start to feed on diatoms.
  • 15. Carbajal-Miranda et al. (2005) Navicula incerta Amphiprora paludosa Haliotis rufescens
  • 16. Daume et al. (2000) Cylindrotheca closterium Haliotis rubra Amphora sp. Navicula sp., N. jeffr Cocconeis sp
  • 17. Gordon et al. (2006) Haliotis discus Amphora luciae Navicula cf. lenzii
  • 18. Haliotis iris Achnanthes longipes Kawamura et al. (1998) Nitzschia ovalis Nitzschia sp Cocconeis p. Navicula britannica
  • 19. Gallardo and Buen (2003) Mixed diatoms vs. monospecific cultures of Navicula Haliotis asinina
  • 20. little information concerning diatoms as feed for Haliotis diversicolor
  • 21. Kawamura et al., 1998 Poor and unpredictable performance of abalone postlarvae differences in lipid, the species of protein and diatoms they extracellular have been fed polymeric substances (EPSs)
  • 22. Hypothetical cellular pathways and processes in iron-limited pennate diatom cells. All roman green or red type depicts gene transcripts found to be up- or down-regulated, respectively. Italicized and underlined green type indicates metabolites found to be enriched relative to total protein in iron-limited cells. DF, diffusion factor; EPS, extracellular polymeric substances; FR, ferric reductase; HMA, heavy metal-associated; PCD, programmed cell death; ROS, reactive oxygen species.
  • 23. AIMS • to improve the growth and the survival of abalone post-larval stages in a specific growth system using specific diatoms species • a better understanding of their basic dietary requirements farmers attempting to produce mollusks and crustaceans from the larval stage • high cost of producing live food (microalgae) • up to 30% of the total cost of production (Valenzuela-Espinoza et al., 1999).
  • 24. Alternative • use immobilized microalgae • can be cheaper than algae produced by traditional methods • ready for use (Chen, 2003).
  • 25. Specific aims • Survey the diversity of diatoms species found in H. diversicolor hatcheries. • Isolate and develop a monoculture that could be immobilized and encapsulated in alginate beads for long-term storage as algal stock.
  • 26. monospecific algal stocks used as feed for cultivating the post-larvae of H. diversicolor • Lipids • Proteins • EPS – soluble EPS – bound EPS – internal carbohydrate – residual carbohydrate
  • 27. Collection and preparatory cultures of benthic diatoms Diatoms from small abalone hatchery ponds scraped from abalone post-larvae settlement substrates (plastic plates) collected in l L collection bottles Yean-Chang Chen, Ph.D. Associate Professor, placed in a cool icebox Department of Aquaculture, National Taiwan Ocean University, transported to the Algal Laboratory Keelung, Taiwan Email: ycchen@mail.ntou.edu. of the National Taiwan Ocean tw b0232@ind.ntou.edu.tw University, Keelung, Taiwan Tel: 886 2 24622192 ext. 5221
  • 28. Martek 120-liter Deep aerated tank used for the culture of Nannochloropsis for photobioreactor aquaculture
  • 29. Eicosapentaenoic acid 20:5(n-3) (EPA, 5,8,11,14,17-cis-eicosapentaenoic acid) EPA is an omega-3 fatty acid • an n-3 C20-polyunsaturated fatty acid that is metabolically active. with a doublefrom the endstartingcarbonthe third carbon atom bond (C=C) of the after chain. a carboxylic acid with a 20-carbon chain and five cis double bonds the first double bond is located at the third carbon from the omega end.
  • 30. EPA and its derivatives have proved beneficial in prevention and treatment of certain medical conditions • coronary heart disease • blood platelet EPA is effective aggregation also in arresting and minimizing • abnormal cholesterol tumor growth levels • several carcinomas
  • 31. EPA is currently sourced from fish oil • No other sources are commercially available. – fluctuates in price and quality. – contamination of fish oil with pesticides and heavy metals. – an alternative economic and consistent source of EPA is needed. – Microalgae are one potential source.
  • 32. Annual EPA demand demand is expected to increase dwindling supplies of fish oil 125 tones in Japan Current market price of EPA ethyl ester (95% pure) about $650/kg