Pallab K. Sarker, Ph.D. 1*, Anne R. Kapuscinski, Ph.D. 1, Alison J. Lanois 1, Erin D. Livesey 1, Katie P. Bernhard 1, Mariah L.

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Towards sustainable aquafeeds: Evaluating three marine microalgae for replacement of

and rainbow trout (Oncorhynchus mykiss). Pallab K. Sarker, Ph.D. 1*, Anne R.

1 Towards sustainable aquafeeds: Evaluating three marine microalgae for replacement of fish oil and fishmeal in aquaculture diets for Nile tilapia (Oreochromis niloticus) and rainbow trout (Oncorhynchus mykiss). Pallab K. Sarker, Ph.D. 1*, Anne R. Kapuscinski, Ph.D. 1, Alison J. Lanois 1, Erin D. Livesey 1, Katie P. Bernhard 1, Mariah L. Coley 1,2 * Pallab Sarker 1 Environmental Studies Program, Dartmouth College 2 Sustainable Agriculture Research and Education Program (SAREP), University of California Davis

FAO 2014 66.6 Mt in 2012 91.3 Mt in 2012 1982 2012 Average increase= 8.6%/year Human health benefits: protein and essential micronutrients.

2 FAO Mt in Mt in Average increase= 8.6%/year Human health benefits: protein and essential micronutrients. Food security: ~ 20% of animal protein intake for ~ 3 billion people. Need +23 million tonnes by 2020 to maintain current consumption rate, 18.6 kg/capita/year. Must come from aquaculture to avoid further ocean depletion. ~ 57% of world s fisheries stock are fully exploited

3 Challenge: use of wild fish in aquafeeds Fishmeal Grinder Fish oil Small ocean fish, healthy human food (Anchovy, sardine, mackerel) Unsustainable: Reduces wild fish, declines marine biodiversity (Troell et al. 2014) Exhaustion of fishmeal and fish oil by 2040 (Duarte et al. 2009) Divert away from human consumption: 50% total food fish supply for people in more than 36 countries (FAO 2012) Net loss of edible fish on a global basis; Fish In: Fish Out: >1.0 (Sarker et al. 2013)

Challenge: Use of wild fish in feeds Fish in, fish out ratio

2 0 Canada Chile Norway Scotland Major Salmon producing

, Forster, I., Were, K., Hicks, B., Vandenberg, G.W., 2013.

4 Challenge: Use of wild fish in feeds Fish in, fish out ratio Canada Chile Norway Scotland Major Salmon producing nations Sarker, P.K., Bureau, D.P., Drew, M., Hua, K., Forster, I., Were, K., Hicks, B., Vandenberg, G.W., Sustainability issues related to feeding salmonids: a Canadian perspective. Reviews in Aquaculture, 5: 1 21.

5 Alternative terrestrial feedstuffs Plant proteins: Soybean meal, canola meal, pea meal, corn gluten, canola/ soy protein concentrate Plant Oil sources: Canola/rapeseed oil, soy oil, flaxseed oil, linseed oil camelina oil

6 Terrestrial crops: Sustainability and Nutrition Question? Unsustainable: Divert away from direct human consumption Driver of unsustainable agricultural practices (Costa Pierce et al. 2012; Foley et al. 2011) Main fatty acid composition of vegetable oils (% of oil) Unbalanced Nutrition: Unbalanced essential amino acid Anti nutritional factors No long chain polyunsaturated fatty acids (omega 3, n3) (Sarker et al. 2013) 18:2n 6, LA 18:3n 3, ALA 20:5n 3, EPA 22:6n 3, DHA Canola Flaxseed Corn Sun flower Soybean Rape Palm Linseed Olive EPA and DHA : Key fatty acids beneficial for human health

Spirulina sp Chlorella sp Schizochytrium sp Nannochloropsis sp Isochrysis sp Why microalgae aquafeed?

meets requirements of fish Why salmonids? Rich source of key fatty acids (EPA and DHA) for human health.

(Shepherd 2011). Why Nile tilapia, Oreochromis niloticus?

7 Spirulina sp Chlorella sp Schizochytrium sp Nannochloropsis sp Isochrysis sp Why microalgae aquafeed? High biomass yield compared to other terrestrial feeds Synthesize health-promoting n3 fatty acids (EPA, DHA) Amino acids profile meets requirements of fish Why salmonids? Rich source of key fatty acids (EPA and DHA) for human health. Priority to meet the sustainability challenge: Salmonids are the largest consumers of the global fish oil (68%) in aquaculture (Shepherd 2011). Why Nile tilapia, Oreochromis niloticus? Globally, 2 nd most farmed fish by volume, production grew ~10%/yr since 1995 (FAO 2012). Feeds low on food chain and evolved to eat algae Significant challenge: Farmed tilapia have depressed n3:n6 ratio (1:6) erodes health benefit of eating tilapia (Weaver et al. 2008)

Quantified digestibility of microalgae in Nile tilapia (Oreochromis niloticus) feed 1 st step to design sustainable diet First study using a marine microalga in Nile tilapia Nile tilapia Fecal Nile

highest digestibility of protein and amino acids good candidate for fish meal replacement in tilapia feed. Chlorella sp. low digestibility due to high fiber content poor candidate for tilapia feed.

8 Quantified digestibility of microalgae in Nile tilapia (Oreochromis niloticus) feed 1 st step to design sustainable diet First study using a marine microalga in Nile tilapia Nile tilapia Fecal Nile collector tilapia Marine Schizochytrium sp. highest digestibility of lipid, fatty acids, and phosphorous; high DHA content good candidate for fish oil replacement in tilapia feed. Spirulina sp. highest digestibility of protein and amino acids good candidate for fish meal replacement in tilapia feed. Chlorella sp. low digestibility due to high fiber content poor candidate for tilapia feed. Sarker, P.K., Gamble, M.M., Kelson, S., Kapuscinski, A.R. (2015). Nile tilapia (Oreochromis niloticus) show high digestibility of lipid and fatty acids from marine Schizochytrium sp. and of protein and essential amino acids from freshwater Spirulina sp. feed ingredients. Aquaculture Nutrition (doi: /anu.12230)

Diet Formulation: Fish oil replacement Objective: Determine the optimum level of fish oil substitution for

75% 100% Ingredient (%) SC0 Sc25 Sc50 Sc75 Sc100 Fish oil 9 6.75 4.5 2.25 0 Schizochytrium sp 0 4 8 12.5 16.

9 Crude protein: 38%; Crude lipid: 10%; Gross energy:16 kj/g Kapuscinski, A.R and Sarker, P.K., 2015.

9 Diet Formulation: Fish oil replacement Objective: Determine the optimum level of fish oil substitution for growth of tilapia and improve deposition of n3 LC PUFAs to rebalance n3:n6 ratio in their fillets 0% 25% 50% 75% 100% Ingredient (%) SC0 Sc25 Sc50 Sc75 Sc100 Fish oil Schizochytrium sp Fish meal Corn gluten meal Others Crude protein: 38%; Crude lipid: 10%; Gross energy:16 kj/g Kapuscinski, A.R and Sarker, P.K., Aquaculture Feed Formulation and Aquaculture Product Produced With the Same. U.S. Provisional Application, DC0562US. L2. Filing date January 23, 2015.

10 Experimental design 5test diets (1 control diet, 4 microalgae diets). 3 replicate tanks per diet. 40 tilapia/tank, fed three times per day. 84 days feeding trial.

Sample collection and analysis Bulk weight every 3 weeks 5 fish/tank for fillet at day 42 (middle) and day 84 (final) Conduct biochemical analyses of diets, individual ingredients, and fillet

11 Sample collection and analysis Bulk weight every 3 weeks 5 fish/tank for fillet at day 42 (middle) and day 84 (final) Conduct biochemical analyses of diets, individual ingredients, and fillet composition Weight gain = final wet weight initial wet weight FCR, feed conversion ratio = feed intake/ weight gain PER, protein efficiency ratio = weight gain (g)/protein fed (g) Survival rate (%) = (Final number of fish / Initial number of fish) x 100

12 Growth indices after 84 days Diet ANOVA Sc0 Sc25 Sc50 Sc75 Sc100 P value Initial weight (g) 1.4 ± ± ± ± ± Final weight (g) 25.3 ± 0.3 bc 26.4 ± 0.4 bc 27.2 ± 0.7 ab 27.4 ± 0.3 ab 28.8 ± 0.2 a <0.01 Weight gain (g) 23.8 ± 0.4 bc 24.9 ± 0.3 bc 25.5 ± 0.7 ab 25.8 ± 0.2 ab 27.3 ± 0.2 a <0.01 FCR 1.1 ± 0.0 bc 1.0 ± 0.0 bc 1.0 ± 0.1 ab 0.9 ± 0.0 ab 0.9 ± 0.0 a <0.01 PER 2.4 ± 0.1 bc 2.6 ± 0.1 bc 2.7 ± 0.2 ab 2.8 ± 0.1 ab 3.1 ± 0.1 a <0.01 Survival rate (%) ± ± ± ± ± Sarker, P.K., A.R. Kapuscinski, A. Lanois, E. Livesey, K. Bernhard and M. Coley Towards sustainable aquafeeds: complete substitution of fish oil with marine microalga Schizochytrium sp. improves weight gain, feed efficiency and health promoting n3:n6 fatty acid ratio in Nile tilapia (Oreochromis niloticus) (PLOS ONE: in review). Patent Pending: Kapuscinski, A.R and Sarker, P.K., Aquaculture Feed Formulation and Aquaculture Product Produced With the Same. U.S. Provisional Application, DC0562US. L2. Filing date January 23, 2015.

13 Relation between diet n3:n6 ratio and fish weight gain Sc100: y = x r = (p<0.01) Weight gain (g) Diet n3:n6 ratio Kapuscinski and Sarker Patent pending; Sarker et al. (PLOS ONE: in review).

4:1 Dried whole cell Sc resulted in significantly higher weight gain (g), feed conversion ratio (FCR) and protein efficiency ratio (PER).

14 DHA and n3:n6 ratio in fillet Provide about 20% of the daily recommended level if a person ate two tilapia fillets (100 g cooked or 130 g raw) per week. mg DHA/100 g fillet a a b b c Sc0 Sc25 SC50 Sc75 Sc100 Fish oil replacement level mg DHA n3:n6= 1.4:1 Dried whole cell Sc resulted in significantly higher weight gain (g), feed conversion ratio (FCR) and protein efficiency ratio (PER). Yielded highest DHA content in fillets and rebalanced low n3:n6 ratio (1:6), common in farmed tilapia, to levels >1:1, more beneficial for human health Kapuscinski and Sarker Patent pending; Sarker et al. (PLOS ONE: in review).

in aquaculture feed Ongoing work: What proportion of fishmeal and fish oil can microalgae/co products replace in aqua feed to reduce dependence on forage fisheries but maintain fish growth?

15 Nannochloropsis sp. and Isochrysis sp. in tilapia & rainbow trout feeds Recent experiments found improved digestibility of whole cells of both microalgae in tilapia and in rainbow trout can be used as substitutes for fishmeal and fish oil in aquaculture feed Ongoing work: What proportion of fishmeal and fish oil can microalgae/co products replace in aqua feed to reduce dependence on forage fisheries but maintain fish growth? Effect on n3 fatty acid content of fish flesh? (experiment underway) How does fish oil and fishmeal replacement by different microalgae/co products affect phosphorous in aquaculture effluents? (Gamble et al. in preparation) Can we achieve total replacement of fish oil and fish meal by combining different marine microalgae/co products in tilapia/trout feeds? (experiment underway) Fertilize microalgae with organic waste streams instead of inorganic fertilizers. Lifecycle effects?

16 Thanks Funding from Dartmouth (Kapuscinski): Sherman Fairchild Professorship in Sustainability Science, and Dean of Arts and Sciences Recently awarded USDA animal nutrition grant (Sarker as lead PI & Kapuscinski as Co PI) Qualitas Health, Inc. Assistants: Michael Perlstein ( 14) and the Fish Girls! Mariah Coley ( 11), Suzanne Kelson ( 12), Madi Gamble ( 13), Tasneem Khalid ( 12), Alison Lanois ( 15), Erin Livesey ( 15), Katie Bernhard ( 15).

OPPORTUNITIES FOR RENDERED PRODUCTS IN AQUACULTURE Advancing science & industry through partnership

OPPORTUNITIES FOR RENDERED PRODUCTS IN AQUACULTURE Advancing science & industry through partnership Jesse T. Trushenski CENTER FOR FISHERIES, AQUACULTURE, & AQUATIC SCIENCES THE WORLD IS HUNGRY CGIAR CCAFS