ABSTRACT Aquafeed manufacturers have reduced, but not fully eliminated, fishmeal and fish oil and are seeking cost competitive replacements. We combined two commercially available

microalgae, to produce a high-performing fish-free feed for Nile tilapia (_Oreochromis niloticus_)—the world’s second largest group of farmed fish. We substituted protein-rich defatted

biomass of _Nannochloropsis oculata_ (leftover after oil extraction for nutraceuticals) for fishmeal and whole cells of docosahexaenoic acid (DHA)-rich _Schizochytrium_ sp. as substitute for

fish oil. We found significantly better (_p_ < 0.05) growth, weight gain, specific growth rate, and best (but not significantly different) feed conversion ratio using the fish-free feed

($1.03/kg tilapia), though the median feed cost ($0.68/kg feed) was slightly greater than that of the reference feed ($0.64/kg feed) (p < 0.05). Our work is a step toward eliminating

reliance on fishmeal and fish oil with evidence of a cost-competitive microalgae-based tilapia feed that improves growth metrics and the nutritional quality of farmed fish. SIMILAR CONTENT

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SUSTAINABLE NUTRITIOUS FOOD PRODUCTION Article Open access 20 November 2023 INTRODUCTION Aquaculture, the world’s most efficient producer of edible protein, continues to grow faster than any

other major food sector in the world, in response to the rapidly increasing global demand for fish and seafood1,2. Feed inputs for aquaculture production represent 40–75% of aquaculture

production costs and are a key market driver for aquaculture production1. The aquafeed market is expected to grow 8–10% per annum and is production of compound feeds is projected to reach

73.15 million tonne (mt) in 20252,3,4,5,6,7,8,9. Ocean-derived fishmeal (FM) and fish oil (FO) in aquafeeds has raised sustainability concerns as the supply of wild marine forage fish will

not meet growing demand and will constrain aquaculture growth1,2,10,11. Moreover, competition for FM and FO from pharmaceuticals, nutraceuticals, and feeds for other animals6,12 further

exacerbates a supply–demand squeeze2,13. The use of forage fish (such as herrings, sardines, and anchovies) for FMFO production also affects human food security because approximately 16.9

million of the 29 mt of forage fish that is caught globally for aquaculture feed is directed away from human consumption every year14. More than 90 percent of these fish are considered food

grade and could be directly consumed by humans, especially food insecure people in developing countries15. Although more prevalent in aquafeeds for high-trophic finfish and crustaceans, FM

and FO is also routinely incorporated (inclusion rates of 3–10%) in aquafeeds for low-trophic finfish like tilapia to enhance growth1,6,16,17,18. Tilapia (dominated by _Oreochromis

niloticus_)—the world’s second top group of aquaculture organisms—is cultured in such large volumes and is such an integral part of human diets across the world, that even low inclusion

rates of FMFO in aquafeeds for this species is a substantial portion of global demand of forage fish (Supplementary Table S1)19. The aquafeed industry reduces reliance on FM and FO by using

grain and oilseed crops (e.g., soy, corn, canola), however, terrestrial plant ingredients have low digestibility, anti-nutritional factors, and deficiencies in essential amino acids (lysine,

methionine, threonine, and tryptophan)16,20. Crop oils also lack long-chain omega-3s (n-3s), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), important for human health21,22.

Elevated levels of n-6 (e.g. linoleic acid) fatty acids from crop oils23,24 changes the long-chain n-3/n-6 ratio in tilapia flesh25 that is passed on to human consumers26,27,28, resulting in

increased production of pro-inflammatory eicosanoids (via arachidonic acid), which has led nutritionists to doubt the health benefits of farmed tilapia21,25. Alternatives to terrestrial

crops have been too costly for broad adoption by aquafeed manufacturers (Sarker et al.15). However, nutritional disadvantages and poor fillet quality have prompted researchers to investigate

marine microalgae as potential FMFO replacements in fish feeds due to balanced essential amino acids, minerals, vitamins, and long-chain n-3 fatty acids17,29,30,31,32,33,34,35,36,37,38. The

peer-reviewed literature, however, lacks information on how using marine microalgae in fish-free diets affects growth, feed conversion and fillet quality of tilapia. There also are limited

published data on the market price of fish-free diets made with alternative ingredients that show potential for economies of scale. We conducted research to develop a new aquafeed formula by

combining the protein-rich (50%) defatted marine microalgal co-products (under-utilized left-over biomass of _Nannochloropsis oculata_ after EPA oil extraction for human supplement) with

another DHA-rich (30% of total fatty acids) marine microalga (_Schizochytrium _sp.), increasingly available at commercial scale, to fully replace FMFO (fish-free) in tilapia aquafeeds. This

study builds on our recent microalgae aquafeeds research. Sarker et al. replaced 33% of FM with under-utilized _N. oculata_ defatted biomass in a tilapia diet that achieved final weight,

weight gain, percent weight gain, specific growth rate, and protein efficiency ratio values comparable to the reference diet containing FM and FO17. Furthermore, it was previously reported

that _Schizochytrium_ sp. is a highly digestible source of nutrients for tilapia and can fully replace FO in tilapia feed30,33. To examine the commercial viability of using marine microalgae

to replace both FM and FO, we conducted a nutritional feeding experiment to compare three microalgal diets to a reference diet containing FM and FO levels found in commercial tilapia feed.

Microalgal diets included defatted _N. oculata_ to replace 33%, 66% or 100% of FM, and whole cell _Schizochytrium _sp. to replace 100% of FO (33NS, 66NS, 100NS). We measured effects of the

four diets on growth metrics, in vitro protein digestibility, feed conversion ratio (FCR), protein efficiency ratio (PER), and fillet deposition of n-3 long-chain polyunsaturated fatty acids

(LC PUFAs) and minerals. Furthermore, we conducted a hedonic analysis to estimate the market price of defatted _N. oculata_ meal and whole cell _Schizochytrium_ sp., feed costs, and the

economic feed conversion ratio (ECR). MATERIALS AND METHODS The experimental design and fish use protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of

Dartmouth College. Also, we conducted all experiments in accordance with relevant guidelines and regulations. We euthanized the fish by single cranial pithing in the nutritional feeding

experiment. DIET FORMULATION FOR NUTRITIONAL FEEDING EXPERIMENT We incorporated _N. oculata _defatted biomass to replace different percentages of FM and whole cell _Schizochytrium _sp. to

replace all FO in three tilapia experimental diets for a nutritional feeding trial. These three diet formulations were based on our previous digestibility data for _N. oculata _defatted

biomass and whole cell _Schizochytrium _sp.17,30,33, and a prior study showing potential to replace all FO with whole cell _Schizyochytrium_ sp.30. We compared these three experimental diets

to a reference diet (served as control diet) containing FMFO at levels found in commercial tilapia feed. All diets were iso-nitrogenous (37% crude protein) and iso-energetic (12 kJ/g).

Microalgae inclusion diets used _N. oculata_ defatted biomass to replace 33% (33NS), 66% (66NS), and 100% (100NS) of the FM and whole cell _Schizochytrium _sp. to replace all FO in the test

diets (33NS, 66NS, 100NS). Thus _N._ _oculata_ comprised 3%, 5% and 8% of the diet by weight, respectively, and _Schizochytrium _sp. made up 3.2% of the diet by weight. We produced the diets

in accordance with our previous work17,30,36. We obtained dried _Schizochytrium_ sp. from ALGAMAC, Aquafauna Bio-marine, Inc., Hawthorne, CA, USA; and menhaden FO from Double Liquid Feed

Service, Inc., Danville, IL, USA. Qualitas Health Inc., which markets EPA-rich oil extracted from _N. oculata_ as a human supplement39 and seeks uses for tons of under-utilized defatted

biomass from its large-scale production facilities, donated the _N. oculata_ defatted biomass. Supplementary Table S8 reports proximate compositions and amino acid profiles of _N. oculata 

_defatted biomass and _Schizochytrium _sp_._; total fatty acid profile by percentage of the defatted biomass and _Schizochytrium _sp ingredients reported in Supplementary Table S9; and

macromineral and trace element composition of both ingredients reported in Supplementary Table S10. The formula, proximate analysis, and amino acid profiles of four dietary treatments

reported in Table 1. The fatty acid profiles reported in Supplementary Table S11 and the macrominerals and trace elements of the four experimental diets reported in Supplementary Table S7.

EXPERIMENTAL DESIGN AND SAMPLING TO EVALUATE TILAPIA GROWTH ON _N. OCULATA_ DEFATTED BIOMASS AND _SCHIZOCHYTRIUM _SP. DIETS We conducted the feeding experiment using a completely randomized

design of four diets × three replicates tanks in recirculating aquaculture systems (RAS). Four hundred eighty Nile tilapia (mean initial weight 34.5 ± 2.06 g) were put into randomized groups

of 40, bulk weighed, and transferred to a tank. Tilapia had been acclimated to the FMFO containing reference diet for 7 days prior to distribution. The initial stocking density remained

within levels recommended to avoid physiological stress on tilapia (< 0.25 lbs/gal in 80 gallon RAS tanks). We carefully monitored water quality daily to maintain favorable conditions for

tilapia across all RAS tanks and kept the water temperature at 28.7 ± 0.25 °C, pH at 7.1 ± 0.1, dissolved oxygen at 6.1 ± 0.15 mg/L, total ammonia nitrogen at 0.26 ± 0.1 mg/L, and nitrite

nitrogen at 0.3 ± 0.01 mg/L17,30. We administered feed at a rate of 8% of body weight until day 60, 6% until day 121, and 4% until day 183, with feedings performed twice per day at 09:00 and

15:30 h. We measured fish biomass monthly by randomly selecting 10 fish as a weight sample to adjust feeding rates for growth and we bulk weighed all fish every other month for sampling

events (day 0, 60, 121, and 185). We withheld feed for 24 h prior to the weighing procedure to reduce handling stress on fish. BIOLOGICAL SAMPLING AND TISSUE COLLECTION We randomly selected

and weighed 10 individual fish from the total starting stock at the beginning of the experiment, then euthanized (by single cranial pithing17, and stored fish tissues at – 20 °C for future

biochemical analysis. At day 121 of the experiment, we euthanized 6 fish per tank, and 6 additional fish at day 185, the terminus of the trial. Half of the fish sampled on day 121 and day

185 were filleted, and half were kept whole and then stored at − 20 °C for further processing17,30. All samples from the initial sampling, day 121, and day 185 were freeze dried at − 20 °C,

then fully homogenized. Both whole body and fillet samples were sent to New Jersey Feed Laboratory, Inc (Ewing, NJ, USA) for full proximate, energy, amino acid, and fatty acid profiles.

ANALYTICAL PROCEDURE AND CALCULATION We quantified final weight, weight gain, weight gain percentage, FCR, SGR, PER, and survival rate for each of the dietary treatments. Each of these

parameters were calculated as follows: weight gain = (final weight − initial weight/initial weight) × 100; FCR, FCR = feed intake/weight gain; protein efficiency ratio; SGR (%/day) = 100 × 

ln final wet weight (g) − ln initial wet weight (g))/Time (days), PER = weight gain (g)/protein fed (g); and survival rate (%) = (final number of fish/initial number of fish) × 10017,34,40.

The trace mineral content of each of the experimental diets, sampled fish fillets, and whole bodies was analyzed by the Department of Earth Science at Dartmouth College17. Each 100 mg sample

was acid digested in 0.5 mL 9:1 HNO3/HCl in open vessel digestion with heating at 105 °C for 1 h. Samples were diluted to 10 mL in DI water prior to analysis. All measurements were recorded

gravimetrically. Digested samples were run by ICP-MS analysis using an Agilent 7700 × with collision (He) and reaction (H2) gases. The methodology and quality control followed EPA method

6020a. DEGREE OF PROTEIN HYDROLYSIS AND IN-VITRO PROTEIN DIGESTIBILITY We performed an in-vitro digestibility assessment according to the method prescribed in Yasumaru and Lemos to measure

the degree of protein hydrolysis of our experimental diets in the presence of tilapia stomach crude enzyme extract and intestine crude enzyme extract41. A 50 g sample from each of the four

diets was ground via mortar and pestle until all materials could fit through a 0.5 mm food sieve. We allotted 80 mg by protein basis of each diet with 25 mL DI water in a 50 mL reaction

vessel immersed in a water bath held at 25 °C. The reaction mixture, containing diet and DI water, was adjusted to pH 2.0 with 0.1 M HCl using a Hannah instrument HI-901C1 potentiometric

auto titrator, set to dose 0.3 mL HCl every 2 min for 30 min until pH equilibrium was reached. After equilibrium, we introduced 200 µL stomach crude enzyme extract prepared according to

Yasumaru and Lemos with storage solution modifications sourced from Chaijaroen and Thongruang41,42. After crude enzyme extract introduction, we made minor pH changes adding 0.1 M HCl or 0.01

 M NaOH by hand when necessary. Once we introduced the crude enzyme extract, we initiated a predetermined program on the auto titrator to dose 0.025–0.075 mL in proportion to the change in

pH measured. This program dosed accordingly every 3-min interval to keep the pH at 2.0 for 1 h. The program was paused, when necessary, to prevent over adjusting the solution during the

titration. After the 1-h stomach digestion period, we recorded the total volume dosed. We then adjusted the reaction mixture pH to 8.0, using 0.1 M NaOH, and allowed the auto titrator to

dose 0.025 mL 0.1 M NaOH for approximately 1 h to allow the mixture to reach equilibrium. Once pH equilibrium was reached, we introduced 250  µL intestinal crude enzyme extract, prepared in

the same way as the stomach crude enzyme extract. Minor adjustments to pH were made by hand using 0.01 M NaOH or 0.1 M HCl. Then we initiated the auto titrator method to dose 0.01–0.025 mL

0.1 M NaOH proportional to the measured change in pH, in order to hold the pH at 8.0 for 1 h, and recorded the total volume dosed. All diets were run in triplicate41,43. We quantified the

degree of protein hydrolysis in the stomach using the following equation: $$DH = \left[ {\frac{V \times N}{E}} \right] \times \left( \frac{1}{P} \right) \times F_{pH} \times 100\% ,$$ (1)

where DH is the degree of hydrolysis, V is the volume of the acid consumed (mL), N is the normality of the acid (H+ available for release × Molarity), E is the mass of the substrate protein

(g), P is the number of peptide bonds cleaved (mol g protein−1) and when amino acid composition is unknown, (8.0), and FpH is the correction factor for pH 2.0 at 25 °C (1.08). We quantified

the degree of protein hydrolysis in the intestine using the following equation: $$DH = B \times Nb \times \left( \frac{1}{a} \right) \times \left( \frac{1}{MP} \right) \times \left(

{\frac{1}{{H_{tot} }}} \right) \times 100\% ,$$ (2) where B is the volume of alkali consumed (mL), Nb is the normality of the alkali (alkali groups × Molarity), a is the average degree of

dissociation of the a-NH2 groups (1/a = 1.50 for pH 8.0 at 25 °C), MP is the mass of substrate protein (g), and Htot is the total number of peptide bonds in the protein substrate [7.6–9.2

meqv g protein−1] according to the source of protein44. After calculating the degree of protein hydrolysis, we determined the in vitro protein digestibility using a prediction equation model

 as reported by Yasumaru and Lemos and Tibbets41,43. The degree of protein hydrolysis was used as input in the following equation to determine in vitro protein digestibility, IPD = (3.5093DH

 + 70.248). ECONOMIC ANALYSIS OF FISH-FREE FEED FORMULATED WITH MICROALGAE BLENDS We obtained commodity and market prices for the formulated feed ingredients from a variety of sources

(Supplementary Tables S5 and S12). We conducted non-parametric bootstraps in RSTUDIO (v.1.2.5033) based on 10,000 replicates using the adjusted bootstrap percentile method to estimate the

median and 95% confidence intervals. We conducted a hedonic analysis in RSTUDIO to estimate the price of defatted _N. oculata_ meal and whole cell _Schizochytrium_ sp. The general

methodology of hedonic analysis is described in Maisashvili et al.45. We used mixed-effects linear models using maximum likelihood methods46,47. Following Maisashvili et al., we selected

crude protein, ether extract, methionine, and lysine as the key input variables in our defatted _N. oculata_ meal model45. We used the following regression formula: $${\varvec{y}}_{t} =

\beta_{0} + b_{{0,CP_{t} }} + b_{{0,EE_{t} }} + \beta_{1} \cdot {\varvec{CP}}^{2} + \beta_{2} \cdot {\varvec{Met}}^{2} + \beta_{3} \cdot {\varvec{Lys}}^{2} + b_{{1_{t} }} \cdot {\varvec{CP}}

+ \left( {\beta_{4} + b_{{2_{t} }} } \right) \cdot {\varvec{EE}} + \varepsilon ,$$ (3) where Yt is the vector of feed ingredient prices observed at time t, CP is a vector of independent

variables reflecting the crude protein content of the corresponding feed ingredients, MET is a vector of independent variables reflecting the methionine content of the corresponding feed

ingredients, LYS is a vector of independent variables reflecting the lysine content of the corresponding feed ingredients, EE is a vector of independent variables reflecting the ether

extract content of the corresponding feed ingredients, β0 is the fixed-effect intercept, β1 is the fixed-effect coefficient of CP2, β2 is the fixed-effect coefficient of MET2, β3 is the

fixed-effect coefficient of LYS2, β4 is the fixed-effect coefficient of EE, b0,CP is the random-effect intercept of CP at time t, b0,EE is the random-effect intercept of EE at time t, b1 is

the random-effect coefficient of CP at time t, b2 is the random-effect coefficient of EE at time t, ε is the residual error, and t is the time period (2010–2019). We selected the top fatty

acids present in both the commodity oils (vegetable and fish) and in _Schizochytrium_ sp. that did not require an extrapolation. Thus, we used the following regression formula:

$${\varvec{y}}_{t} = \beta_{0} + b_{{0,14:0_{t} }} + b_{{0,16:0_{t} }} + \beta_{1} \cdot {\user2{20:5}}{\mathbf{n}}{ - }{\user2{3}}^{2} + \beta_{2} \cdot {\user2{14:0}}^{2} + \beta_{3} \cdot

{\user2{16:1}}{\varvec{n}}{ - }{\user2{7}}^{2} + \left( {\beta_{4} + b_{{1_{t} }} } \right) \cdot {\user2{14:0}} + \left( {\beta_{5} + b_{{2_{t} }} } \right) \cdot {\user2{16:0}} +

\varepsilon ,$$ (4) where Yt is the vector of oil ingredient prices observed at time t, 20:5N-3 is a vector of independent variables reflecting the EPA content of the corresponding oil

ingredients, 14:0 is a vector of independent variables reflecting the myristic acid content of the corresponding oil ingredients, 16:1N-7 is a vector of independent variables reflecting the

palmitoleic acid content of the corresponding oil ingredients, 16:0 is a vector of independent variables reflecting the palmitic acid content of the corresponding oil ingredients, β0 is the

fixed-effect intercept, β1 is the fixed-effect coefficient of 20:5N-32, β2 is the fixed-effect coefficient of 14:02, β3 is the fixed-effect coefficient of 16:1N-72, β4 is the fixed-effect

coefficient of 14:0, β5 is the fixed-effect coefficient of 16:0, b0,14:0 is the random-effect intercept of 14:0 at time t, b0,16:0 is the random-effect intercept of 16:0 at time t, b1 is the

random-effect coefficient of 14:0 at time t, b2 is the random-effect coefficient of 16:0 at time t, ε is the residual error, and t is the time period (2010–2019). As inputs to Eqs. (3) and

(4), we used the mean annual prices for 12 meal ingredients and 7 oil ingredients from January 2010 to December 2019 (see Supplementary Table S12 for details about the commodities and data

sources). Although some studies have used shorter time horizons for their hedonic models (e.g. 2 years)48, we followed other studies that used longer time horizons (e.g. 10 years) in their

hedonic models49 and economic analysis of agricultural commodities to capture variability50. We incorporated a freight component to calculate the costs to bring these commodities to the Port

of Shanghai, China. To account for the multi-modal components of the freight costs of U.S. commodities, we applied modal transport shares (e.g. rail, truck, barge) of grain commodities

(e.g. corn, wheat, soybeans, sorghum, and barley) to the distances between the grain production sites and U.S. ports (see Supplementary Table S13 and Supplementary Methods for further

details). We used a shipping route distance calculator to estimate the international shipping distances (Supplementary Table S14). We obtained the nutritional composition of the feed

commodities from Archer Daniel Midlands and Feedinamics (Supplementary Table S15). We obtained the fatty acid profiles of the oils used in the feed from the literature (Supplementary Table

S16). For the terrestrial-plant-based oils, we used the fatty acid values reported in Dubois et al.51. For FO, we used the fatty acid values reported in Sarker et al.30. We scaled the

vectors of independent variables (Supplementary Tables S15 and S16) with the parameters provided in Supplementary Tables S17 and S18, for defatted _N. oculata_ and whole cell

_Schizochytrium_ sp., respectively. We assessed the goodness of fit using graphical methods and diagnostic tests (see Supplementary Methods, Supplementary Tables S19 and S20, and

Supplementary Figs. S2–S7 for further details). We estimated the price of defatted _N. oculata_ meal with Eq. (3), the scaled parameters (Supplementary Table S21), the fixed-effect

coefficients (Supplementary Table S22), and the random-effect coefficients (Supplementary Table S23). We estimated the price of whole cell _Schizochytrium_ sp. with Eq. (4), the scaled

parameters (Supplementary Table S24), the fixed-effect coefficients (Supplementary Table S25), and the random-effect coefficients (Supplementary Table S26). To convert the estimated price of

_Schizochytrium_ sp. oil to whole cell _Schizochytrium_ sp., we multiplied the price by the fraction of lipids in _Schizochytrium_ (0.54). We calculated the costs of all ingredients of

formulated reference feed and experimental feeds (which combined _N. oculata_ defatted biomass with _Schizochytrium _sp.) to determine the diet costs in USD per kg (Supplementary Table S27).

The price of each diet was determined by multiplying the respective contributions of each feed ingredient by their respective costs per kg and summing the values obtained for all of the

ingredients in each of the formulated diets. Finally, we estimated the production cost of tilapia ($/kg fish) via ECR to compare among the four experimental tilapia feeds (which combined 

defatted biomass with _Schizochytrium_ sp.). We estimated fish production cost as ECR using the equation of Piedecausa52: $$ECR \left( {\frac{\$ }{{{\text{kg}}\,fish}}} \right) = FCR \left(

{\frac{{{\text{kg}}\, diet\, fed}}{{{\text{kg}} \,weight \,gain}}} \right) \times price \,of\, diet \left( {\frac{USD\$ }{{{\text{kg}} \,diet}}} \right),$$ (5) where ECR is the economic

conversion ratio, and FCR is the feed conversion ratio. STATISTICAL ANALYSIS Statistical analysis (ANOVA) was performed according to Sarker et al.17 to determine the significant differences

in proximate and amino acid content, fatty acid profile, final weight, weight gain, weight gain percentage, in vitro protein digestibility, FCR, SGR, PER, survival rate, and ECR for each of

the treatments. When significant differences were found, we compared the treatment means using Tukey’s test of multiple comparisons (posthoc), with a 95% confidence interval. The IBM

Statistical Package for the Social Sciences (SPSS) program for Windows (v. 21.0, Armonk, NY, USA) was used for all statistical methods. DATA AND CODE AVAILABILITY The datasets and RSTUDIO

files used in the economic analysis including the hedonic regression analyses (used to estimate the price of defatted _N. oculata_ meal and whole cell _Schizochytrium_), bootstrap confidence

intervals of feed ingredient prices, and the ECR for Fig. 2 are available at the following link: https://doi.org/10.6071/M3VD5V. RESULTS GROWTH, NUTRIENT UTILIZATION AND PROXIMATE

COMPOSITION OF TILAPIA CARCASS Fish fed the fish-free diet for 184 days displayed significantly better (_p_ < 0.05) final weight, weight gain, percent weight gain and specific growth rate

than fish fed the reference diet, which contained FM and FO levels typically found in commercial tilapia diets (Table 2). Growth rates were linear throughout the experiment and weights

measured for the fish-free diet diverged from those for the reference diet by day 128 (Supplementary Fig. S1). Tilapia fed fish-free feed showed an improved food conversion ratio and protein

use efficiency ratio though differences among diets and were not statistically significant. We detected no difference in survival rate among all diets and all fish appeared healthy (no

visual signs of illness or deformities) at the end of the experiment. The whole-body proximate composition (Supplementary Table S2) did not significantly differ across the dietary

treatments; lipid contents ranged from 2 to 5% and protein contents ranged from 13 to 17% across the four treatments. FILLET PROXIMATE AND AMINO ACID COMPOSITION We detected the highest

crude protein, lipid, and ash content in the fillet tissue of tilapia fed the fish-free feed (100NS), with the only significant difference (_p_ < 0.05) being crude lipid (Supplementary

Table S3). Crude protein contents ranged from 18–24% among the four dietary treatments. Nile tilapia fillets from the fish-free feed treatment had significantly higher lipid content (1.8%)

compared to fillets from the reference (0.8%), 33NS, (0.9%), and 66NS (0.9%) feeds. The fillet amino acid composition, except for methionine and histidine, did not differ across the diets

(Supplementary Table S4). We detected significantly lower (_p_ < 0.05) methionine and histidine content in the 33NS diet compared to other diets. Methionine and histidine content in the

66NS diet was the highest when compared to the fish-free and reference diets, but was not significantly different. FILLET MACRO MINERALS AND TRACE ELEMENTS COMPOSITION We did not find any

significant differences in macromineral composition in fillets across all diets (Table 3). Fillet trace element composition also did not significantly differ across the dietary treatments,

except for selenium, which differed significantly (_p_ < 0.05) between the reference and 33NS diets but not among the reference, fish-free and 66 NS diets. We detected the lowest level of

arsenic in fish fillet of fish-free feed. Other trace elements—boron, mercury, lead and molybdenum—were at non-detectable levels in all fish fillets. FILLET FATTY ACID (% OF TOTAL FATTY

ACIDS) CONTENT The fillet of tilapia fed the experimental diets was similar to the dietary fatty acid content of the corresponding feed. Across diets, the concentrations of total n-3 PUFA,

n-6 PUFA, n-3 LC PUFA, and n-6 LC PUFA, were not significantly different (Table 4). We also found that the total saturated fatty acid (SFA), most of the SFA fractions, total mono-unsaturated

fatty acids (MUFA), and most MUFA fractions did not differ across diets. Fish fed the reference diet displayed the highest (_p_ < 0.05) concentrations of 16:1n-7 which corresponds to the

16:1n-7 content in experimental diets. In the fillet of fish fed the reference and fish-free feed, we detected similar MUFA fractions of 16:1n-9, 18:1n-7, and 20:1n-9. Total PUFAs were

significantly higher (_p_ < 0.05) in tilapia fillet fed microalgae inclusion diets (33NS, 66NS, and 100NS) compared to the reference diet. Many of the individual PUFAs did not vary

greatly among dietary treatments. However, n-6 fatty acids, 18:3n-6, 20:3n-6, 22:4n-6, and 22:5n-6 showed significant differences (_p_ < 0.05) between the diets. Among n-3 PUFAs, we

detected significantly higher (_p_ < 0.05) 22:6n-3 DHA in tilapia fed microalgae inclusion diets compared to reference diet. The highest EPA content in the reference diet reflected the

higher EPA supplied by this diet. The reference diets had the highest concentrations of 20:3n-6, and 22:4n-6 compare to the three other treatments. In contrast, tilapia fed the reference

diet had significantly (_p_ < 0.05) decreased concentrations of 22:5n-6 compared to fish fed microalgae inclusion diets. The n-3/n-6 PUFA ratios did not differ significantly between all

four dietary treatments. The n-3/n-6 LC PUFA ratio was highest in the fish-free and reference diets. AMOUNTS OF MAJOR N-3 AND N-6 PUFA (MG/G) IN THE FILLET The amount of n-3 PUFAs, EPA and

DHA did differ among diets (Supplementary Table S11). All diets that combined _Schizochytrium_ with _N. oculata _defatted biomass enhanced the DHA deposition in the fillet. Tilapia fed

fish-free feed, 100NS diet deposited a significantly higher (_p_ < 0.05) amount of DHA (5.15 mg/g) than fish fed the reference diet which deposited DHA at 2.47 mg/g (Fig. 1). The EPA

content of fish fed the reference diet was significantly higher (_p_ < 0.05) compared to the other three diets and reflected the higher EPA supplied by this diet. The amounts of major n-6

PUFA deposition in the fish fillet (mg/g fillet) were not significantly different among diets. DEGREE OF PROTEIN HYDROLYSIS AND IN-VITRO PROTEIN DIGESTIBILITY We detected the highest degree

of protein hydrolysis and in-vitro protein digestibility in the fish-free feed (100NS), although the difference was not statistically significant compared to the reference feed (Table 5).

ECONOMIC ANALYSIS OF FISH-FREE FEED FORMULATED WITH MICROALGAE BLENDS Here, we compared the estimated ingredient prices, the formulated feed prices and the ECR across experimental diets

formulated with microalgae blends and the reference diet. Results of the hedonic regression analysis show that the median price [and 95% confidence interval] is $0.44 [0.39, 0.49] and $2.38

[1.93, 2.57] per kg biomass for defatted _N. oculata_ and whole cell _Schizochytrium_ sp., respectively (Supplementary Table S5). While the median price of soybean meal is modestly greater

(1.07 times) than the median price of defatted _N. oculata_, the median price of FM is nearly 3.5 times the median price of defatted _N. oculata_. In contrast to defatted _N. oculata_ being

much cheaper than FM, the median price of whole cell _Schizochytrium_ sp. is roughly 1.4 times the median price of FO. Owing to this greater price of _Schizochytrium_ sp. compared with FO,

the median [and 95% confidence interval] price of the fish-free feed that combined defatted _N. oculata_ meal with whole cell _Schizochytrium_ sp. (100NS), at $0.68 [0.62, 0.73] per kg feed,

was slightly greater than the reference diet at $0.64 [0.61, 0.68] per kg feed (Table 6). The ECR, defined as the price of the formulated feed in US dollars per kg tilapia weight gain, of

the fish-free feed was smaller than ECR of the reference diet (Fig. 2 and Table 6), despite the slightly greater price of the fish-free feed (100NS) compared to reference diet. We detected

significant differences (_p_ < 0.05) in ECR across all diets. While not significantly different, the ECR of the fish-free feed (100NS) at $0.95 [0.90, 0.98]/kg tilapia was roughly 92% the

ECR of the reference diet ($1.03 [1.00, 1.07]/kg tilapia) (Fig. 2). This can be explained by the smaller FCR of the fish-free feed (1.40 ± 0.06) compared with reference diet (1.61 ± 0.05).

DISCUSSION Our results demonstrate the feasibility of combining commercially available microalgal biomasses to formulate fish-free aquaculture feeds that are high-performing and show

potential to become cost-competitive. This is the first report of successfully combining protein-rich-defatted biomass of one microalgal species with DHA-rich whole-cell biomass of another

microalgal species to achieve full replacement of FM and FO ingredients in a tilapia feed formulation. This also is the first report of improved feed utilization metrics, including growth,

weight gain, specific growth rate, and of beneficial DHA fatty acid profile in Nile tilapia fed a fish-free microalgal diet compared to a commercial feed formulation containing FM and FO.

Production is increasing for both types of microalgal biomass used in the fish-free diet, indicating good potential to achieve economies of scale. Our estimate of the ECR for the fish-free

diet supports the proposition that biomass from these microalgae will inevitably become cost competitive with FM and FO commodities. NUTRITIONAL BENEFIT OF COMBINING _N. OCULATA_ DEFATTED

BIOMASS AND _SCHIZOCHYTRIUM_ IN THE FISH-FREE DIET The combination of _Schizochytrium_ sp. and defatted biomass of _N. oculata_ in the fish-free feed exhibited two major benefits. First,

fish fed the fish-free feed had improved growth consistent with our prior observations that _Schizochytrium_ sp. is a highly digestible ingredient for tilapia33 and that elevated levels of

_Schizochytirum_ sp. led to improved growth, FCR, and PER30. Second, we found the highest in-vitro protein digestibility in the fish-free feed, suggesting that protein originating from

defatted _N. oculata_ biomass was the most digestible when in the presence of highly digestible _Schizochytrium_ sp., presumably due to the latter triggering certain digestive enzymes,

release and activity. Thus, the combination of defatted _N. oculata_ biomass and _Schizochytrium_ sp. appears to be better suited to the digestive enzymes present in tilapia digestive

systems than conventional diets with FMFO; and the presence of _Schizochytrium_ sp. may support more efficient digestion of the fish free-feed at the higher inclusion levels of _N. oculata_ 

defatted biomass. However, further research is necessary to elucidate the digestive enzyme profiles present under different dietary regimes and to assess the differences in the digestibility

of microalgal fish-free feeds compared to conventional feed with FMFO. Other studies also point to benefits of including _Schizochytrium_ in aquafeeds. Our prior study reported better

digestibility, improved growth, fillet protein, and lipid content by Nile tilapia fed diets with inclusion of _Schizochytrium_ in fish-free feed. Similar results were reported in a study

that found dietary inclusion of _Schizochytrium_ sp. stimulated muscle or tissue development of Atlantic salmon53. Our observations of beneficial effects of including _Schizochytrium_ in

fish-free feed on the growth of tilapia is also consistent with findings in shrimp and barramundi, which demonstrated an algal derived DHA stimulated growth performance54,55. Moreover, high

levels of a micronutrient, such as the carotenoid, astaxanthin, and bioactive compounds, in DHA-rich _Schizochytrium_ could contribute to the growth of fish30,55. Significantly lower weight

gain of tilapia fed the reference feed compared to fish-free feed also seems consistent with the fact the FM and FO in reference diet had limited dietary 22:6n-3 DHA. This would cause

increased energy expenditure for de novo DHA biosynthesis, given that DHA biosynthesis is a rather expensive metabolic exercise. Such diversion of energy to DHA biosynthesis would reduce the

growth performance of tilapia. The human health benefit of using highly digestible 22:6n-3 DHA-rich _Schizochytrium_ is reflected in this study, given that tilapia fed the fish-free feed

yielded the highest amount of 22:6n-3 DHA in fillet—almost twice that of conventional feed (Supplementary Table S6). Results are consistent with our previous findings where increasing levels

of _Schizochytrium _sp. corresponded to reduced levels of FO in tilapia feed and resulted in significant increases in fillet 22:6n-3 DHA deposition compared to a reference diet containing

FMFO30. Nile tilapia is not an oily fish like salmon, but nevertheless deserves efforts to improve nutritional value of farmed fish because it is produced in huge tonnages and is an

important component of human diets in many parts of the world, especially Asia and Africa. Thus, improvement of tilapia nutritional value through increased levels of DHA could benefit a very

large number of people, many of whom have low levels of n-3 LC-PUFA in their diets23. Our results support the relative ease of enhancing the n-3 LC PUFA composition of tilapia fillets,

while also achieving a fish-free diet, by combining _Schizochytrium _sp. and _N. oculata_ defatted biomass. Tilapia with elevated DHA levels after eating fish-free feed will have tremendous

market potential56. Feed manufacturers can exploit this feature to market aquafeeds to aquaculturists aiming to cater to health-conscious consumers who are willing to pay a premium for

DHA-enhanced tilapia fillets. Tilapia fed reference feed exhibited significantly increased amounts of 20:5n-3 EPA compared to microalgae-inclusion diets due to a higher concentration of

20:5n-3 EPA in the reference diet. Our results on fillet deposition of ALA, EPA and DHA can be explained by prior research and the relative abundance of these fatty acids in _Schizochytrium 

_sp. IMPACTS OF FISH-FREE DIET ON MACROMINERALS AND TRACE ELEMENTS The literature has little data on the elemental composition of microalgae; and we found that most of the essential

macrominerals and trace elements were at higher levels in _N. oculata_ defatted biomass and _Scizochytrium_ sp whole cells (Table 3) than in conventional terrestrial feed ingredients57. We

found higher levels for most macrominerals in the _Scizochytrium_ sp whole cells than _N. oculata_ defatted biomass, and higher levels of trace elements in the _N. oculata_ defatted biomass

than in _Scizochytrium_ sp whole cells (Table 3). Depositions of macrominerals and several trace elements in tilapia fillet were not significantly different among all dietary treatments

(Table 3). We found non-detectable levels of boron, mercury, and lead in tilapia fillets across all diets. Moreover, most of the trace element concentration in fillet was lower than the

concentration of all experimental diets. We previously suggested that these trace elements may be excreted and less absorbed by Nile tilapia58,59. We detected the lowest level (0.03 mg kg−1)

of total arsenic in the fish-free microalgae feeds and the highest level (0.33 mg kg−1) in reference feed (Supplementary Table S7). However, the level of total arsenic in all the diets

(0.03–0.33 mg kg−1) including reference feed was below the European Union level of 10 mg kg−1 set for in aquaculture feed60. High levels of arsenic have been previously reported in FOs, thus

contributing considerably to higher arsenic levels in commercial aquaculture feeds61,62,63. The level of total arsenic in the fillet of tilapia did not differ across the diets (Table 3),

and the levels were in the range between 0.14-0.21 mg kg−1 lower than reported values in Atlantic salmon fillet (0.3–1.1 mg kg−1)64. FEED CONVERSION RATIO (FCR) CONSIDERATIONS FCR is a key

driver of farming efficiency, economic and environmental performance. Improving the FCR of farmed tilapia through improved feed technology would help increase the cost effectiveness of

fish-free diets. Tilapia farming can further reduce the FCR close to 1:1 by a variety of means including better feed formulations using highly digestible feed ingredients, use of appropriate

pellet size for each life stage, and better on-farm feed management practices (e.g., storage and feeding rates). Extruded sinking pelleted feed could improve overall FCR; moreover,

extrusion or enzymatic processing of under-utilized, defatted biomass of microalgae, such as _N. oculata_ used in this study, could further improve the FCR of fish-free feed, and also help

push feed formulated with microalgae towards being cost-competitive with conventional feed17,65. ECONOMIC ANALYSIS OF FISH-FREE FEED FORMULATED WITH MICROALGAE BLENDS Our estimate of the

market price of defatted _N. oculata_ meal is in good agreement with another study that used hedonic methods to estimate the of market price of defatted _N. oculata_ meal45. However, key

differences between our study and the study conducted by Maisashvili et al. is that we used more recent commodity prices (January 2010 to December 2019 instead of January 2005 to December

2012) and the list of commodities used in our analysis are more representative of tilapia feed ingredients instead of ingredients for carnivorous fish and shrimp feed. With respect to whole

cell _Schizochytrium_ sp., we are unaware of other studies using hedonic methods to estimate the implied market price of this ingredient. Nevertheless, our implied price results for whole

cell _Schizochytrium_ sp. are in general agreement with studies that have used alternative methods66,67. The similar estimated costs of the fish-free feed (100NS) and reference diet suggest

that using combinations of microalgal biomass, that are on track to achieve economies of scale, is a feasible strategy for achieving large-scale production of cost-competitive fish-free

diets. An emerging path to economies of scale for the two microalgae used in this study is a biorefinery business model whereby oil rich fractions of the microalgal biomass are marketed as

high-value products, such as omega-3 rich human supplements, and other fractions as lower-priced feed ingredients68,69. _N. oculata_ contains an appreciable amount of the omega-3 fatty acid,

EPA70. The projected global growth of over 14% in omega-3 fatty acids from microalgae in the near future will result in a large supply of defatted biomass67. Furthermore, the production of

_Schizochytrium_ sp., already at commercial-scale, is also anticipated to grow, as the projected compound annual growth rate of DHA from microalgae sources is expected to exceed 10% in the

near future67. In order for such high-performing fish-free feed for tilapia to succeed in the market, we acknowledge that _Schizochytrium_ sp. needs to become cost-competitive with FO

sources for aquaculture feeds. Analysts predict ongoing technological improvements and R&D efforts to produce _Schizochytrium_ sp. will quickly make it a cost competitive substitute for

FO due to lower production costs and higher market availability71,72. FO substitutes with _Schizochytrium_ sp have emerged within the last year with new products from many agribusiness

giants and animal nutrition companies (Corbion, BioMar, Archer Daniels Midland and Veramaris), presumably due to favorable economics and high production volumes. A commercial producer of 

_Schizochytrium _oil, Veramis, recently joined a global challenge to sell the most “fish-free” oil for aquafeed to reduce demand pressures on wild-caught stocks, the fish-free feed (F3) FO

Challenge73. Alternative feed ingredients like natural marine algal oil have also recently been approved for use in the supply chain by the UK retailer, Tesco74. Given the proliferation of

alternative feed ingredients by global industry leaders and stakeholders (aquafeed company, innovators, aquafarmers, investors, and aquaculture supply chain), market opportunities appear to

be growing and evolving for using microalgal protein and oil for fish-free feed75,76. CONCLUSION Our results provide a framework for the development of fish-free feeds and the first evidence

of a high performing feed for tilapia that combines two different marine microalgae. Defatted marine microalgae, a protein-rich biomass left over after extracting oil for other products, is

currently under-utilized (often creating disposal problems even though it is food-grade), and is increasingly available as the algal-oil nutraceutical market grows. Advancing the use of

microalgal defatted biomass in aquafeeds would improve the sustainability of aquaculture by reducing its reliance on FM extracted from forage fisheries. Combining under-utilized defatted

biomass protein with DHA-rich marine microalga in the fish-free feed resulted in better tilapia growth compared with fish fed a conventional diet containing FMFO. Furthermore, tilapia fed

the fish-free feed yielded the highest amount of DHA in the fillet, almost twice higher than in those fed conventional feed. Thus, feeding a DHA-rich, microalgae blended diet to farmed

tilapia is a practical way to improve human health benefits of eating farmed tilapia. Moreover, these results suggest other kinds of microalgae combinations are possible and worthy of future

investigation. Our fish-free formulation also shows potential cost-competitiveness, given that the ECR of the fish-free diet was slightly lower, though not significantly different, than the

reference diet. The microalgal ingredients in our fish-free feed, thus, show potential to supply the expanding aquaculture industry with a stable and affordable supply of healthy protein

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provided by Agriculture and Food Research Initiative Competitive Grant no. 2016-67015-24619 from the USDA NIFA (to PKS); Dartmouth College from the Sherman Fairchild Professorship (to ARK),

Dean of the Faculty, and Vranos family gift; University of California Santa Cruz, Dean of Social Sciences and Executive Vice Chancellor; and the National Sea Grant Aquaculture Federal

Funding Opportunity, Social, Behavioral and Economic Research Needs in Aquaculture (NOAA-OAR-SG-2019-2005953). We thank Qualitas Health, Inc. for donating under-utilized _Nannochloropsis

oculata_ defatted biomass for this research. We thank the Department of Earth Sciences, Dartmouth for conducting analytical chemistry (macro minerals and trace elements). AUTHOR INFORMATION

AUTHORS AND AFFILIATIONS * Environmental Studies Department, University of California Santa Cruz, Santa Cruz, CA, 95064, USA Pallab K. Sarker, Anne R. Kapuscinski, Brandi McKuin, Devin S.

Fitzgerald & Connor Greenwood * Health Professions Program, Sciences, Mathematics and Biotechnology, University of California Berkeley Extension, 1995 University Ave., Suite 200,

Berkley, CA, 94704-7000, USA Hannah M. Nash Authors * Pallab K. Sarker View author publications You can also search for this author inPubMed Google Scholar * Anne R. Kapuscinski View author

publications You can also search for this author inPubMed Google Scholar * Brandi McKuin View author publications You can also search for this author inPubMed Google Scholar * Devin S.

Fitzgerald View author publications You can also search for this author inPubMed Google Scholar * Hannah M. Nash View author publications You can also search for this author inPubMed Google

Scholar * Connor Greenwood View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Conceived and designed the experiments: P.K.S., A.R.K.;

performed the experiments: D.S.F., H.M.N., P.K.S.; analyzed the data: P.K.S., B.M., C.G.; contributed reagents/materials/analysis tools: P.K.S., D.S.F., B.M., C.G.; wrote- original draft:

P.K.S.; wrote- review and editing: P.K.S., A.R.K., B.M. CORRESPONDING AUTHOR Correspondence to Pallab K. Sarker. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Sarker, P.K., Kapuscinski, A.R., McKuin, B. _et al._ Microalgae-blend tilapia feed eliminates fishmeal and fish oil, improves growth, and is

cost viable. _Sci Rep_ 10, 19328 (2020). https://doi.org/10.1038/s41598-020-75289-x Download citation * Received: 12 June 2020 * Accepted: 12 October 2020 * Published: 12 November 2020 *

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