PROTEIN-RICH BIOMASS OF THRAUSTOCHYTRIDS, CULTURING METHOD AND USES

Abstract

The invention is in the field of cultures of microalgae, in particular thraustochytrids. The invention relates to a protein-rich biomass of thraustochytrids, to a method of obtaining said biomass, and to the uses thereof.

Claims

1. A thraustochytrid biomass comprising at least 35% by weight of proteins relative to the weight of dry matter.

2. The biomass according to claim 1, wherein it comprises at least 45% proteins.

3. The biomass according to claim 1, wherein it comprises at least 60% proteins.

4. The biomass according to claim 1, wherein it comprises less than 20% fat by weight relative to the weight of dry matter.

5. The biomass according to claim 4, wherein it comprises less than 10% fat.

6. The biomass according to claim 1, wherein said thraustochytrids are of a genus selected from the group consisting of Aurantiochytrium, Aplanochytrium, Botryochytrium, Japonochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium and Ulkenia.

7. The biomass according to claim 6, wherein said thraustochytrids are selected from the species Aurantiochytrium mangrovei CCAP 4062/2; Aurantiochytrium mangrovei CCAP 4062/3; Aurantiochytrium mangrovei CCAP 4062/4; Aurantiochytrium mangrovei, CCAP 4062/5; Aurantiochytrium mangrovei CCAP 4062/6; Aurantiochytrium mangrovei CCAP 4062/1; Schizochytrium sp. 4087/3; Schizochytrium sp. CCAP 4087/1; Schizochytrium sp. CCAP 4087/4; Schizochytrium sp. CCAP 4087/5.

8. The biomass according to claim 1, wherein it has a moisture content of 1% to 95%.

9. The biomass according to claim 8, wherein it has a moisture content of 70% to 90%.

10. The biomass according to claim 8, wherein it has a moisture content of 1% to 10%.

11. A process for producing a biomass as defined in claim 1, wherein it comprises: a. a first step of culturing thraustochytrids in a defined culture medium comprising a carbon source, a nitrogen source, a phosphorus source and salts, said first step a) being divided into two sub-steps, a first sub-step: a1) of growth in the suitable culture medium until carbon source contents in the medium lower than 20 g/L are obtained, followed by a second sub-step: a2) of production wherein one or more carbon source, nitrogen source and/or phosphorus source enrichment solutions are added to the culture medium, simultaneously or successively, until a culture density of at least 40 g/L dry matter is obtained; b. a second step of recovering the biomass obtained in the first step by separating said biomass from the culture medium.

12. The process according to claim 11, wherein in step a2) the carbon source content is maintained between 0 and 50 g/L, the nitrogen source content is maintained between 0.5 and 5 g/L and the phosphorous source content is maintained between 0.5 and 5 g/L.

13. The process according to claim 11, wherein it comprises a third step: c. of drying the biomass recovered in the second step.

14-15. (canceled)

16. A method for improving animal performance comprising administering to the animal the thraustochytrid biomass as described in claim 1.

17. The method according to claim 16, wherein the improvement in performance is evaluated by measuring consumption, weight gain or feed conversion ratio.

18. A cosmetic or pharmaceutical composition for humans or animals comprising a biomass as described in claim 1.

19. A food product, wherein it comprises a biomass as described in claim 1.

20. A livestock feed, wherein it comprises 1% to 60% of a biomass as described according to claim 1.

21. The feed according to claim 20, wherein it comprises 1% to 20% of the said biomass.

22. The feed according to claim 21, wherein it comprises 3% to 8% of the said biomass.

23. A livestock feed, wherein it comprises 1% to 40% of a biomass according to claim 1.

24. The feed according to claim 23, wherein it comprises 5% to 10% of the said biomass.

25. (canceled)

Description

[0107] The invention also has as an object the use of the biomass as described above in therapy, as well as in the prevention and treatment of malnutrition.

[0108] FIG. 1: Experimental design of the measurement of apparent metabolizable energy (AME) in 23-day-old chickens; (test 14ALG069)

[0109] FIG. 2: Experimental design of the choice and consumption test in chicks (7 and 9 days old); (test 14ALG081)

[0110] FIG. 3: Measurement of feed consumption at 7 days of age under choice conditions (corn-soybean feed vs feed substituted with 5%, 10% and 15% of the microalga tested) (test 14ALG081)

[0111] FIG. 4: Measurement of feed consumption at 9 days of age under choice conditions (corn-soybean feed vs feed substituted with 5%, 10% and 15% of the microalga tested) (test 14ALG081)

[0112] FIG. 5: Measurement of mean relative feed consumption over the 2 tests (at 7 d and 9 d) under choice conditions (corn-soybean feed vs feed substituted with 5%, 10% and 15% of the microalga tested) (test 14ALG081)

[0113] FIG. 6: Measurement of growth performance (consumption, weight gain, feed conversion ratio) of 0- to 7-day-old chicks fed a control corn-soybean diet vs feed substituted with 5%, 10% and 15% of the microalga tested (not under choice conditions) (test 14ALG081)

[0114] FIG. 7: Measurement of growth performance (consumption, weight gain, feed conversion ratio) of 0- to 9-day-old chicks fed a control corn-soybean diet vs feed substituted with 5%, 10% and 15% of the microalga tested (not under choice conditions) (test 14ALG081)

EXAMPLES

[0115] Other aspects and features of the invention will become apparent upon reading the following examples and in the appended figures describing same.

[0116] FIG. 1 shows the experimental design of the measurement of apparent metabolizable energy (AME) in 23-day-old chickens; (test 14ALG069). Approximately 300 1-day-old male chicks (Ross PM3) were placed in the same low-density enclosure and fed a standard starter containing wheat, corn and soybean meal. At 13 days of age the chickens are starved for 2 hours before being weighed and distributed by weight group. One hundred twenty chickens are thus selected, placed in individual metabolic cages and assigned to one of the experimental treatments according to their weight (20 repetitions per diet).

[0117] Feed and water are distributed ad libitum throughout the test. The experimental feeds are provided as 3.2-mm-diameter pellets. After a 7-day period of adaptation to the diets and the metabolic cages, a total excreta collection period, flanked by 17-hour fasts, was carried out for 3 days, from day 20 to day 23. The excreta are collected individually each day, combined and stored at ?20? C. At the end of the test the collected excreta are freeze-dried then left at room temperature for a water-uptake phase (48 h) in order to stabilize the moisture content before weighing, grinding (0.5 mm) and analyses.

[0118] FIG. 2 shows the experimental design of the choice and consumption tests in chicks (7 and 9 days old); (test 14ALG081). Two experimental tests were carried out in parallel.

[0119] 1) Test 1choice test: For the test with choice of feed, approximately 200 1-day-old male chicks (Ross PM3) were placed in groups of about 20 in divided metabolic cages and were fed a standard starter containing wheat, corn and soybean meal. At 6 days of age the chickens are starved for 2 hours before being weighed and distributed by weight group. One hundred twenty chickens are thus selected, placed by groups of 4 into 30 divided metabolic cages and assigned at day 7 to one of the experimental treatments according to their weight (10 repetitions per treatment). Each cage contains two feeding dishes containing different feeds, corresponding to the following three experimental treatments: [0120] Treatment 1: corn-soybean starter (feeding dish 1) and 5% microalga (feeding dish 2) [0121] Treatment 2: corn-soybean starter (feeding dish 1) and 10% microalga (feeding dish 2) [0122] Treatment 3: corn-soybean starter (feeding dish 1) and 15% microalga (feeding dish 2)

[0123] Feed and water are dislributed ad libitum throughout the test. The experimental feeds are provided as 3.2-mm-diameter pellets. Consumption is measured at T.sub.0+1h, T.sub.0+2h, T.sub.0+3h, T.sub.0+4h and T.sub.0+6h at 7 and 9 days of age, with the feeding dishes in the cages being switched every hour. Between the two consumption measurements (day 8), the animals receive the wheat-corn-soybean starter.

[0124] 2) Test 2consumption measurement: in a second test, the chicks have access to only one type of feed, optionally supplemented with microalga (FIG. 6). Approximately 400 1-day-old male chicks (Ross PM3) were weighed and distributed by weight group. Two hundred forty selected animals are placed by groups of 4 at day 1 in 60 undivided cages in blocks of homogeneous weight (15 blocks of 4 cages) with one feeding dish per cage, each with an experimental feed (15 repetitions per feed). In each cage, the 4 chicks are identified individually. Feed and water are distributed ad libitum throughout the test. The experimental feeds are corn-soybean starter supplemented with 0%, 5%, 10% or 15% microalga, also used in test 1 with choice of feed. Individual live weights are measured at 1, 7 and 9 days of age.

[0125] Uneaten feeds are weighed and consumption per cage is measured at 7 and 9 days of age. From 0 to 7 days of age, the animals fed the feed containing 10% microalga have a significantly improved weight gain compared to the control and to the other two supplemented diets.

[0126] FIG. 3 shows the measurement of feed consumption at 7 days of age under choice conditions (corn-soybean feed vs feed substituted with 5%, 10% and 15% of the microalga tested) (test 14ALG081);

[0127] FIG. 4 shows the measurement of feed consumption at 9 days of age under choice conditions (corn-soybean feed vs feed substituted with 5%, 10% and 15% of the microalga tested) (test 14ALG081);

[0128] FIG. 5 shows the measurement of mean relative food consumption over the 2 tests (at 7 d and 9 d) under choice conditions (corn-soybean feed vs feed substituted with 5%, 10% and 15% of the microalga tested) (test 14ALG081);

[0129] FIG. 6 shows the measurement of growth performance (consumption, weight gain, feed conversion ratio) of 0- to 7-day-old chicks fed a control corn-soybean diet vs feed substituted with 5%, 10% and 15% of the microalga tested (not under choice conditions) (test 14ALG081);

[0130] FIG. 7 shows the measurement of growth performance (consumption, weight gain, feed conversion ratio) of 0- to 9-day-old chicks fed a control corn-soybean diet vs feed substituted with 5%, 10% and 15% of the microalga tested (not under choice conditions) (test 14ALG081).

Example 1

Strain Cultures

[0131] Strain Precultures:

[0132] An Aurantiochytrium mangrovei preculture is prepared on a shaker table (140 rpm) in a temperature-controlled enclosure (26? C.), in preculture medium, containing 4 g of yeast extract as nitrogen source, and 30 g of glucose as carbon source. After 48 hours of incubation the cells are centrifuged for 5 minutes at 3000 g and the cell pellet is rinsed with preculture medium containing neither yeast extract nor any other source of mineral or organic nitrogen. The purpose of this operation is to avoid any supply of Na.sup.+ in the main culture via the addition of yeast extract. The preculture corresponds to 1/100 (v/v) of the culture volume of the main solution. In the case of strain Schizochytrium sp. CCAP 4062/3,27 g/L of NaCl is added to this medium.

[0133] Culture Monitoring:

[0134] Total biomass concentration is monitored by measuring the dry mass (filtration on GF/F filter, Whatman, then oven drying, at 105? C., for at least 24 h before weighing).

[0135] Analyses of total lipid and FAME contents are carried out according to the methods classically described in the literature (Folch et al., A simple method for the isolation and purification of total lipids from animal tissues. Folch J. et al., J Biol Chem. 1957 May; 226(1):497-509.; Yokoyama et al., Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. November; Mycoscience, 2007 Vol. 48, pp. 199-211).

Example 1.1

Comparative

[0136] Aurantiochytrium mangrovei CCAP 4062/5 cultures are prepared in 1- to 2-L fermenters (bioreactors) for use with computer-controlled automated systems. The composition of the culture medium and those of the addition solutions are as follows:

TABLE-US-00001 Main solution Concentration (g/L) KCl 0.36 H.sub.3BO.sub.3 0.175 MgSO.sub.47H.sub.2O 6.750 CaCl.sub.22H.sub.2O 0.55 KNO.sub.3 0.04667 KH.sub.2PO.sub.47H.sub.2O 0.30940 Na.sub.2EDTA2H.sub.2O 3.094 .Math. 10.sup.?3 ZnSO.sub.47H.sub.2O 7.3 .Math. 10.sup.?5 CoCl.sub.26H.sub.2O 1.6 .Math. 10.sup.?5 MnCl.sub.24H.sub.2O 5.4 .Math. 10.sup.?4 Na.sub.2MoO.sub.42H.sub.2O 1.48 .Math. 10.sup.?6 Na.sub.2SeO.sub.3 1.73 .Math. 10.sup.?6 NiSO.sub.46H.sub.2O 2.98 .Math. 10.sup.?6 CuSO.sub.45H.sub.2O 9.8 .Math. 10.sup.?6 EDTA-Fe 0.03 Carbon g/L Glucose 55 Nitrogen g/L (NH4).sub.2SO.sub.4 7 Addition after autoclave Vitamins g/L Thiamin 8.0 .Math. 10.sup.?3 Vitamin B12 1.3 .Math. 10.sup.?4 Pantothenate 2.7 .Math. 10.sup.?3

TABLE-US-00002 Addition solution 1 Concentration (g/L) K.sub.2SO.sub.4 31.9 MgSO.sub.47H.sub.2O 25.8 KH.sub.2PO.sub.47H.sub.2O 61.38 FeSO.sub.47H.sub.2O 0.61 (NH.sub.4).sub.2SO.sub.4* 138.24 MnCl.sub.24H.sub.2O 0.165 ZnSO.sub.47H.sub.2O 0.165 CoCl.sub.26H.sub.2O 1.6 .Math. 10.sup.?3 Na.sub.2MoO.sub.42H.sub.2O 1.6 .Math. 10.sup.?3 CuSO.sub.45H.sub.2O 0.11 NiSO.sub.46H.sub.2O 8.6 .Math. 10.sup.?2 Na.sub.2EDTA2H.sub.2O 1.81 Thiamin 0.49 Vitamin B12 8.0 .Math. 10.sup.?2 Pantothenate 0.1656 Addition solution 2 Concentration g/L Glucose 750 KH.sub.2PO.sub.4 6.4 (NH.sub.4)2SO.sub.4* 34

[0137] The system is adjusted to pH 6 by adding base (NaOH, or KOH) and/or acid (sulfuric acid solution). The culture temperature was set to 26? C. Dissolved oxygen pressure was regulated in the medium throughout the culture, by shaking speed (250-1200 rpm), air flow rate (0.25-1 vvm), or oxygen flow rate (0.1-0.5 vvm). The regulatory parameters, integrated into the controller, made it possible to maintain a constant pO.sub.2 of 5% to 30%. Culture time was 20 to 100 hours, preferably 25 to 96 hours, for example 50 hours.

[0138] During the culture, three additions of addition solution 1 were carried out, as well as additions of solution 2 in order to maintain glucose concentrations between 200 mM and 500 mM.

Example 1.2

Invention

[0139] Aurantiochytrium mangrovei CCAP 4062/5 cultures are prepared in fermenters as essentially described in Example 1.1.

[0140] The procedure is modified in terms of the mode of pH regulation by addition of ammonia (NH.sub.4OH) to avoid the large supply of Na+or K+related to pH regulation by sodium hydroxide or potassium hydroxide, and which may pose problems in terms of the development of animal feed. Part of the nitrogen necessary for culturing the cells is supplied via the regulation of pH by ammonia (NH.sub.4OH).

[0141] The medium used for this example is described in Table 1 below. Unlike the medium described in Example 1.1, this chemically defined medium makes it possible to sustain growth throughout the culture without nutritional limitations.

TABLE-US-00003 TABLE 1 Concentration (g/L) Magnesium chloride 1.08 .Math. 10.sup.?2 Calcium chloride 0.55 Cobalt chloride hexahydrate (CoCl.sub.26H.sub.2O) 1.08 .Math. 10.sup.?4 Manganese(II) chloride tetrahydrate 1.08 .Math. 10.sup.?2 Magnesium sulfate heptahydrate 8.01 Zinc sulfate heptahydrate 1.08 .Math. 10.sup.?2 Nickel sulfate hexahydrate 5.60 .Math. 10.sup.?3 Copper sulfate pentahydrate 7.20 .Math. 10.sup.?3 Potassium sulfate 2.09 Iron sulfate heptahydrate 4.00 .Math. 10.sup.?2 Boric acid 1.75 .Math. 10.sup.?2 Ethylenediaminetetraacetic acid disodium dihydrate 0.12 Sodium dihydrate molybdate 1.08 .Math. 10.sup.?4 Sodium selenite 1.73 .Math. 10.sup.?7 Thiamin 3.20 .Math. 10.sup.?2 Cobalamin or vitamin B12 5.20 .Math. 10.sup.?4 Pantothenate or vitamin B5 1.08 .Math. 10.sup.?2 Glucose (carbon source) 55.00 Ammonium sulfate (nitrogen source) 9.00 Potassium dihydrogen phosphate (phosphorus 4.00 source) Defoamer 0.40

[0142] The physicochemical parameters are controlled during the culture by means of integrated regulators, with pH maintained around a setting of 5, temperature set to 30? C., and pO.sub.2 maintained around a setting of 30% until maximum shaking and air flow rate values are reached.

[0143] During the culture, additions of addition medium, as described in Table 2 below, are carried out so as to maintain the glucose concentration in the culture medium between 20 g/L and 50 g/L.

TABLE-US-00004 TABLE 2 Glucose (carbon source) 688 g/L Ammonium sulfate (nitrogen source) 76 g/L Potassium dihydrogen phosphate (phosphorus source) 39 g/L

Example 1.3

Invention

[0144] Aurantiochytrium mangrovei CCAP 4062/5 cultures are prepared in fermenters under culture conditions as essentially described in Example 1.1.

[0145] The procedure is modified in terms of the media and the mode of addition thereof.

[0146] The cultures are started in batch mode on the medium presented in Table 3 below.

TABLE-US-00005 TABLE 3 Initial concentrtion Compound in the medium Glucose + Glucose1H.sub.2O 55.000 g/L Sea salt Sea salt 15.000 g/L Mineral MgSO.sub.47H.sub.2O 1.683 g/L salts K.sub.2SO.sub.4 2.080 g/L KH.sub.2PO.sub.4 4.000 g/L MnCl.sub.24H.sub.2O 10.800 mg/L ZnSO.sub.47H.sub.2O 1.080 mg/L CoCl.sub.26H.sub.2O 0.011 mg/L Na.sub.2MoO.sub.42H.sub.2O 0.108 mg/L CuSO.sub.45H.sub.2O 0.720 mg/L NiSO.sub.46H.sub.2O 0.560 mg/L FeSO.sub.47H.sub.2O 40.000 mg/L Vitamins Thiamin 32.000 mg/L Vitamin B.sub.12 0.520 mg/L Pantothenate 10.800 mg/L Nitrogen (NH.sub.4).sub.2SO.sub.4 9.000 g/L EDTA Na.sub.2EDTA 0.096 g/L
The continuous culture mode is then started once the biomass reaches 20 g/L in the medium. The feed solution supplied continuously is that described in Table 4 below.

TABLE-US-00006 TABLE 4 Compound Feed solution (g/L) Glucose1H.sub.2O 110.000 Sea salt 15.000 MgSO.sub.47H.sub.2O 4.959 K.sub.2SO.sub.4 6.129 KH.sub.2PO.sub.4 11.786 MnCl.sub.24H.sub.2O 3.182 .Math. 10.sup.?2 ZnSO.sub.47H.sub.2O 3.182 .Math. 10.sup.?2 CoCl.sub.26H.sub.2O 3.2 .Math. 10.sup.?4 Na.sub.2MoO.sub.42H.sub.2O 3.200 .Math. 10.sup.?4 CuSO.sub.45H.sub.2O 2.121 .Math. 10.sup.?2 NiSO.sub.46H.sub.2O 1.650 .Math. 10.sup.?2 FeSO.sub.47H.sub.2O 1.179 .Math. 10.sup.?1 Thiamin 9.429 .Math. 10.sup.?2 Vitamin B12 1.530 .Math. 10.sup.?3 Pantothenate 3.182 .Math. 10.sup.?2 (NH.sub.4).sub.2SO.sub.4 26.518 EDTA 4.092 .Math. 10.sup.?1

[0147] Preliminary tests showed that the dilution rate used directly effects the protein content of the biomass (results not shown). For this example, the dilution rate used is 0.13 h.sup.?1, which corresponds to half the maximum growth rate of the strain under the culture conditions used.

Example 1.4

Comparative

[0148] Schizochytrium sp. CCAP 4062/3 cultures are prepared in fermenters as essentially described in Example 1.1, with culture medium supplemented with 27 g/L and 35.6 g/L NaCl for the initial fermenter medium and the addition medium, respectively.

Example 1.5

Invention

[0149] Schizochytrium sp. CCAP 4062/3 cultures are prepared in fermenters as essentially described in Example 1.2, with culture medium supplemented with 27 g/L and 35.6 g/L NaCl for the initial fermenter medium and the addition medium, respectively.

[0150] Results

TABLE-US-00007 DM (g/L) AA/DM (%) Fat/DM (%) Soybean (beans)* 40.43 21.80 Soybean (meal)* 48.80 0.55 Example 1.1 (comparative) 90.0 30.00 34.30 Example 1.2 (invention) 67.0 57.90 6.10 Example 1.3 (invention) 60.1 35.23 6.56 Example 1.4 (comparative) 87.0 20.44 45.97 Example 1.5 (invention) 79.9 49.66 13.6 DM: Dry matter; AA: Amino acids; *Values in % relative to 100% DM according to the values given by the USDA

[0151] It is noted that the culture processes of the art intended for the production of fats enriched in polyunsaturated fatty acids do not make it possible to obtain compositions with a high protein content (expressed in wt % of amino acids relative to dry matter).

Example 2

Animal Nutrition

Example 2.1

Materials and Methods

[0152] Strain Aurantiochytrium Mangrovei CC4062/5 was cultivated under the conditions of Example 1.2. These conditions are those used in the following examples unless otherwise mentioned.

[0153] 2.1.1Biomass Composition:

[0154] The microalgal biomass was first analyzed for its proximate composition ((moisture (EAU-H internal method adapted to Regulation EC 152/2009 of 27 Jan. 2009 (103? C./4h)SN), fats by hydrolysis (Regulation EC 152/2009 of 27 Jan. 2009Procedure BSN), crude proteins estimated on the basis of the sum of the total amino acid concentrations (Directive 98/64/EC, 9/3/99Standard NF EN ISO 13903), mineral substances (Regulation EC 152/2009 of 27 Jan. 2009SN)), and gross energy (NF EN 14918), total fiber (AOAC 985.29SN), insoluble walls (according to the method of Carre and Brillouet (1989, Determination of water-insoluble cell walls in feeds: interlaboratory study. Journal Association of Official Analytical Chemists, 72, 463-467), calcium (NF EN ISO 6869March 2002CT), phosphorus (Regulation EC 152/2009 of 27 Jan. 2009CT), chloride (internal method adapted from ISO 1841-2July 1996SN), potassium (internal methodICP 05/07SN), sodium (internal methodICP 05/07SN) and total amino acid concentrations.

[0155] 2.1.2Measurement of Amino Acid Digestibility in Caecectomized Cockerels:

[0156] In parallel, this microalga is evaluated in vivo for the measurement of amino acid digestibility in caecectomized cockerels (Green et al., 1987). The animals (adult Isa Brown cockerels) were force-fed a mash composed of one of the ingredients tested (microalga tested or a control soybean meal) and supplemented with wheat starch to obtain a protein level of 18% corresponding to the animals' need. Twelve cockerels housed in individual cages were used per treatment. Each animal received on average 171.2?14.5 g of mash after being starved for 24 h. The excreta collected up to 48 h after force-feeding were collected by group of 4 cockerels such that 3 excreta per treatment are freeze-dried. They were then left at room temperature for a water-uptake phase (48 h) in order to stabilize the moisture content, then weighed, ground (0.5 mm) and analyzed by the Terpstra method (Terpstra and de Hart, 1974) for their non-uric acid nitrogen content and for their total amino acid concentrations (JEOL AminoTac JLC-500N). Endogenous losses of amino acids were previously determined by evaluating a protein-free diet based on the same experimental design. The corrected results of the basal endogenous losses are thus expressed as a standardized ileal digestibility coefficient of amino acids, calculated and subjected to statistical analysis as follows:

[00001]${\mathrm{DIS}?\left(X\right)}_F=\left[\frac{X_F*I_D*L_F-X_E*Q_E+\frac{X_{\mathrm{END}}*I_D}{10}}{X_F*I_D*L_F}\right]*1000$

with DIS (X).sub.F: standardized ileal digestibility coefficient (%) of amino acid X of raw material F; X.sub.F: concentration (g/100 g) of the amino acid in the raw material; X.sub.E: concentration (g/100 g) of the amino acid in the excreta; I.sub.D: amount of feed consumed (g); Q.sub.E: amount excreted (g); L.sub.F: incorporation ratio of the raw material in the feed; X.sub.END: endogenous losses of amino acid X (g/100 g).

[0157] The experimental results are first analyzed for ingredient effect according to an ANOVA procedure (XLSTAT 2010.4.02? Addinsoft 1995-2010), according to the following model with a 95% confidence interval:

Y.sub.i?+a.sub.i

[0158] with: [0159] Y=parameter [0160] ?=mean [0161] a.sub.i=ingredient effect

[0162] For each significant difference, the means are analyzed by a pairwise Fisher's LSD test.

Example 2.2

Composition of Strain CC4062/5 Cultivated for 49 h

[0163] Strain CC4062/5 was cultivated as mentioned above. The culture was stopped at 49 h, producing several kilograms of microalgal biomass for the purpose of carrying out a preliminary digestibility test in the animals. This biomass was dried on a drying cylinder and has the form of flakes. It is identified as strain 4, 49-h culture.

[0164] The biochemical composition results for the microalga, strain 4, 49-h culture are presented in Table 5 below and are expressed as both gross and corrected dry matter (DM) content.

TABLE-US-00008 TABLE 5 (test 14ALG058): Composition of the microalga tested (strain 4, 49-h culture) vs a control soybean meal Concentrations Concentrations (gross %) (% of dry matter) Soybean Soybean ?A meal ?A meal Dry matter, % 94.8 89.5 Moisture, % 5.2 10.5 Sum of amino acids 50.8 46.9 53.4 52.4 Fats 10.6 5.0 11.2 5.6 Crude fiber n/d 4.2 n/d 4.7 Mineral substances 7.3 5.8 7.7 6.5 Starch <1.0 n/a <1.1 n/a Total sugars 1.3 n/d 1.4 n/d ADF n/d 3.7 n/d 4.1 NDF n/d 7.2 n/d 8.0 Total dietary fiber (TDF) 16.5 n/d 17.4 n/d Water-insoluble cell 4.9 n/d 5.2 n/d wall (WICW) Calcium 0.118 0.371 0.124 0.414 Phosphorus 1.448 0.598 1.527 0.668 Chloride (Cl.sup.?) 0.150 n/d 0.158 n/d Potassium 1.983 n/d 2.092 n/d Sodium 0.068 n/d 0.072 n/d (kcal/kg) (kcal/kg DM) Gross energy 4870 4486 5137 5012 n/d: not determined n/a: not applicable ?A: microalga

[0165] These results show that the microalga has a total nitrogenous matter (sum of amino acids) and mineral substances composition slightly higher than that of the control soybean meal. The mineral content of the biomass tested is comparable to the lowest concentrations published, ranging from 7% to 43% DM depending on the family, genus and species of the microalga. The phosphorus and calcium contents are inversely proportional with 2.3 times more phosphorus and 3.3 times less calcium in the microalga relative to the control soybean meal. The strain 4, 49-h culture tested does not contain starch reserves and its total sugar concentration remains low (1.4% DM) compared to the raw materials conventionally used in animal nutrition. In contrast, its fat content is two times higher than that of soybean meal (11.2% vs 5.6% DM) and its gross energy 125 kcal/kg DM higher.

[0166] Microalgae walls have different compositions and structures than those of raw materials of plant origin, and render unsuitable the fiber analyses conventionally used. Also, analysis of total dietary fiber (TDF) (Prosky et al., 1988) has the advantage of being less restrictive and the microalga, strain 4, 49-h culture has a total fiber percentage of 17.4% DM (which, by way of comparison, is about what is analyzed in rye or barley grain) with a water-insoluble cell wall (WICW) residue measured at 5.2% DM.

[0167] The total protein concentrations reported in Tables 4 to 8 derive from the sum of amino acids themselves assayed according to the method described above. The sum of amino acidsas estimator of the total proteins of the microalga, strain 4, 49-h culturecorresponds to 53.4% DM, with more precisely 21.5% DM of essential amino acids and 32.0% DM of nonessential amino acids. Table 6 shows the amino acid concentrations analyzed in the microalga and the soybean meal and expressed as gross values or relative to the sum of amino acids.

[0168] The results reflect that the microalga has a relatively balanced amino acid profile, which agrees with the work comparing the amino acid profiles of different microalgae with those of so-called conventional protein sources (Becker, 2007).

[0169] Generally, the essential amino acid concentrations of the microalga are slightly higher than those measured in the soybean meal (21.5% DM and 24.0% DM, respectively).

TABLE-US-00009 TABLE 6 (test 14ALG058): Total nitrogen and amino acid concentrations (gross %), amino acid concentrations relative to the sum of amino acids (% sum AA), standardized ileal digestibility coefficients of amino acids (%) measured in caecectomized cockerels, and digestible amino acid concentrations (gross %) of the microalga tested vs a control soybean meal. Standardized ileal Digestible [AA] AA/sum AA digestibility of AA [AA] (gross %) (%) (%) (gross %) ?A SM ?A SM ?A SM p-value ?A SM Dry matter 94.8 89.5 Total nitrogen 10.6 7.8 80.1 ? 0.3 89.6 ? 0.7 <0.0001 8.49 6.97 Sum AA 50.8 46.9 78.4 ? 1.0 88.5 ? 1.0 0.001 39.79 41.53 Essential AA 20.4 21.5 Methionine 0.85 0.64 1.67 1.36 85.6 ? 1.3 90.9 ? 1.6 0.023 0.73 0.58 Lysine 2.30 2.87 4.53 6.12 84.9 ? 1.3 87.6 ? 0.8 0.068 1.95 2.51 Threonine 1.71 1.88 3.37 4.01 80.7 ? 1.8 86.0 ? 1.7 0.038 1.38 1.62 Tryptophan 0.55 0.67 1.08 1.43 82.9 ? 1.7 90.3 ? 1.3 0.009 0.46 0.61 Arginine 6.83 3.42 13.44 7.29 61.0 ? 0.8 93.2 ? 0.8 <0.0001 4.17 3.19 Isoleucine 1.48 2.30 2.91 4.90 85.4 ? 1.2 88.7 ? 1.2 0.048 1.26 2.04 Leucine 2.52 3.69 4.96 7.87 86.7 ? 1.1 88.5 ? 1.2 0.182 2.18 3.27 Valine 2.01 2.35 3.96 5.01 87.5 ? 1.6 89.0 ? 1.5 0.389 1.76 2.09 Histidine 0.66 1.18 1.30 2.52 88.7 ? 0.6 89.9 ? 1.4 0.327 0.59 1.06 Phenylalanine 1.52 2.48 2.99 5.29 84.6 ? 1.5 89.8 ? 1.0 0.014 1.29 2.23 Nonessential AA 30.3 25.5 Cystine 0.55 0.63 1.08 1.34 60.9 ? 3.7 79.7 ? 2.0 0.003 0.34 0.50 Serine 1.87 2.52 3.68 5.37 76.7 ? 1.6 88.5 ? 1.3 0.001 1.43 2.23 Proline 1.07 2.14 2.11 4.56 84.3 ? 2.1 87.9 ? 1.3 0.107 0.90 1.88 Alanine 2.17 2.07 4.27 4.41 84.9 ? 1.1 84.9 ? 1.1 0.992 1.84 1.76 Aspartic ac. 3.57 5.49 7.03 11.71 n/d 85.9 ? 0.6 n/d n/d 4.71 Glutamic ac. 18.10 8.81 35.63 18.78 73.4 ? 0.7 90.4 ? 0.7 0.0001 13.29 7.96 Glycine 1.81 2.01 3.56 4.29 78.3 ? 2.3 84.3 ? 1.6 0.036 1.42 1.69 Tyrosine 1.18 1.78 2.32 3.80 84.2 ? 1.7 89.5 ? 0.6 0.015 0.99 1.59 ?A: microalga; SM: soybean meal; [AA]: amino acid concentration

Example 2.3

Measurement of Biomass Amino Acid Digestibility (Strain CC4062/5 Cultivated for 49 h)

[0170] The results concerning standardized ileal digestibility (SID) of amino acids measured in caecectomized cockerels, as well as the digestible amino acid concentrations, are presented in Table 6. The digestibilities of nitrogen and the sum of amino acids of the microalga are 80.1?0.3% and 78.4?1.0%, respectively, which are 9.5 and 10.1 points lower than the control soybean meal. The arginine and cystine SIDs of the microalga have the lowest results, respectively 61.0?0.8% and 60.9 35 3.7%. Generally, the digestibility coefficients of the essential amino acids of the microalga range from 80.7?1.8% (threonine SID) to 88.7?0.6% (histidine SID). Lysine digestibility is measured at 85.6?1.3%. These coefficients are about 1.2 (histidine SID) to 7.4 (tryptophan SID) points lower than those measured for the control soybean meal. However, they reflect high digestibility coefficients, making it possible to consider the evaluated microalga as a protein source of good quality (i.e., they are significantly higher than the microalgae protein digestibility values reported in the literature by about 55% to 77% (Henman et al., 2012).

Example 2.4

Composition of Strain CC4062/5 Cultivated for 22 h

[0171] Strain CC4062/5 was cultivated under the same conditions but for a period of 22 h. It is identified as strain 4, 22-h culture.

[0172] As before, this biomass was dried on a drying cylinder and has the physical form of flakes.

[0173] It was analyzed for its composition of proximates, gross energy, total fiber, insoluble walls, calcium, phosphorus, chloride, potassium, sodium and total amino acid concentrations.

[0174] The biochemical composition of this microalga, strain 4, 22-h culture is presented in Table 7. The results are expressed as both gross and corrected dry matter (DM) content.

[0175] The microalga, strain 4, 22-h culture has a total nitrogenous matter composition (sum of amino acids) 2.3 and 1.3 points higher than those of the control soybean meal and the microalga, strain 4, 49-h culture.

[0176] Its mineral substance content increases relative to a longer culture but remains at a concentration level which is in the lower range of the values published in the literature (Skrede et al., 2011).

[0177] The phosphorus/calcium ratio is unbalanced. The phosphorus and calcium contents are inversely proportional with 3.6 times more phosphorus and 3.6 times less calcium in the microalga relative to the control soybean meal. The optimized strain 4 (22-h culture) contains 1.5 times more total phosphorus than during a 49-h culture. The fat content is higher than that of the soybean meal (8% vs 5.6% DM), and the results confirm that neither starch nor total sugars represent a form of energy storage in this strain 4. Gross energy is measured at 4641 kcal/kg DM, or 371 kcal/kg DM less vs the control soybean meal.

[0178] The total dietary fiber (TDF) of the microalga, strain 4, 22-h culture represents 12.7% of DM with a water-insoluble cell wall (WICW) residue measured at 2.1% DM. In parallel, the residue deduced as follows: R (%)=100mineral substances (%)total nitrogenous matter (%)fat (%)starch (%)sugars (%) indicates that the TDF content is 15.1 points lower than that of R thus estimated at 27.8% DM. These observations agree with other work (Lieve et al., 2012) reporting about 20% to 30% of the amount of the dried biomass not explained by the sum of the contents of mineral substances, lipids, proteins and carbohydrates.

[0179] The microalga, strain 4, 22-h culture has 2 points higher fat than the soybean meal taken in comparison (Table 7).

TABLE-US-00010 TABLE 7 (test 14ALG065): Composition of the microalga tested (strain 4, 22-h culture) vs a control soybean meal AA concentrations AA concentrations (gross %) (% dry matter) Soybean Soybean ?A meal ?A meal Dry matter 94.6 89.5 Moisture 5.4 10.5 Sum of amino acids 51.7 46.9 54.7 52.4 Fat 7.6 5.0 8.0 5.6 Crude fiber n/d 4.2 n/d 4.7 Mineral substances 9.0 5.8 9.5 6.5 Starch <0.5 n/a <0.5 <0.6 Total sugars <0.5 n/d <0.5 <0.6 ADF n/d 3.7 n/d 4.1 NDF n/d 7.2 n/d 8.0 Total dietary fiber (TDF) 12.0 n/d 12.7 n/d Water-insoluble cell 2.0 n/d 2.1 n/d wall (WICW) Calcium 0.109 0.371 0.115 0.415 Phosphorus 2.279 0.598 2.410 0.668 Chloride (Cl.sup.?) 0.160 n/d 0.169 n/d Potassium 2.238 n/d 2.366 n/d Sodium 0.161 n/d 0.170 n/d (kcal/kg) (kcal/kg DM) Gross energy 4390 4486 4641 5012 n/d: not determined; n/a: not applicable ?A: microalga

[0180] The sum of amino acids amounts to 54.7% of DM, with more precisely 23.9% DM of essential amino acids and 30.8% DM of nonessential amino acids. Table 8 presents the amino acid concentrations analyzed in the microalga and the soybean meal and expressed as gross values or relative to the sum of amino acids. The results reflect that the microalga has a relatively balanced amino acid profile compared to the control soybean meal. The glutamic acid, arginine and aspartic acid of the microalga, strain 4, 22-h culture are present in higher proportions with values (% sum AA) of 26.75%, 9.48% and 8.26%, respectively. The lysine content (% sum AA) is 5.94% in reference to that of the control soybean meal (6.12%). Arginine, methionine and, to a lesser extent, threonine contribute more strongly to the microalga protein tested than to that of the soybean meal (9.48% vs 7.29%, 2.13% vs 1.36%, and 4.27% vs 4.01%, respectively). The microalga cultivated for 22 h has 2.4 points more essential acids than a 49-h culture, or the same content as that of the control soybean meal. Except for arginine, essential amino acids are present in a larger proportion of the sum of amino acids.

[0181] The microalga, strain 4, 22-h culture has methionine, arginine and threonine contents higher than those of the soybean, whereas the lysine and valine contents are equivalent or slightly higher in the microalgal biomass.

Example 2.5

Measurement of Biomass Amino Acid Digestibility (Strain CC4062/5 Cultivated for 22 h)

[0182] The measurement of amino acid digestibility in caecectomized cockerels (Green et al., 1987) was performed identically to that described above (see Example 2.3).

[0183] The results concerning standardized ileal digestibility (SID) of amino acids of the microalga, strain 4, 22-h culture measured in caecectomized cockerels, as well as the digestible amino acid concentrations, are presented in Table 8. The digestibilities of nitrogen and the sum of amino acids of the microalga are 85.7 ?0.7% and 87.1 ?0.4%, respectively, or 3.2 (p=0.008) and 0.9 (p=0.190) points lower than the control soybean meal. Compared to the 49-h culture, the digestibility of the sum of amino acids of this production of microalga, strain 4, 22-h culture is significantly increased by 8.7 points. The concentrations of digestible lysine, methionine, arginine, threonine and valine are higher than those of the control soybean meal. They reflect a good quality of the protein of the microalga tested, superior or equivalent to that of soybean meal.

[0184] The results of measurement of amino acid digestibility in cockerels show that the microalga, strain 4, 22-h culture is a protein source of good quality. The digestibility of the proteins (sum of amino acids) is similar to that of soybean meal, which is the protein source most used worldwide for feeding monogastrics.

[0185] Furthermore, the drying process used does not appear to be a factor that denatures the quality of the protein, given the high digestibility coefficients measured for lysine.

[0186] It is important to note that the quality of the protein is significantly improved when the results of the microalga, strain 4, 49-h culture are compared with those of the microalga, strain 4, 22-h culture. Notably, protein digestibility increases by about 9 points.

TABLE-US-00011 TABLE 8 (test 14ALG065): Total nitrogen and amino acid concentrations (gross %), amino acid concentrations relative to the sum of amino acids (% sum AA), standardized ileal digestibility coefficients of amino acids (%) measured in caecectomized cockerels, and digestible amino acid concentrations (gross %) of the microalga tested vs a control soybean meal. Standardized ileal Digestible [AA] AA digestibility of AA [AA] (gross %) (%) (%) (gross %) ?A SM ?A SM ?A SM p-value ?A SM Dry matter 94.6 89.5 Total nitrogen 11.5 7.8 85.7 ? 0.7 88.9 ? 0.5 0.008 9.86 6.92 Sum AA 51.7 46.9 87.1 ? 0.4 88.0 ? 0.7 0.190 45.06 41.2 Essential AA 22.6 21.5 Methionine 1.10 0.64 2.13 1.36 90.0 ? 0.4 91.5 ? 0.3 0.015 0.99 0.59 Lysine 3.07 2.87 5.94 6.12 87.9 ? 0.2 85.6 ? 0.3 0.0001 2.70 2.46 Threonine 2.21 1.88 4.27 4.01 87.3 ? 0.3 85.2 ? 1.0 0.045 1.93 1.60 Tryptophan 0.68 0.67 1.32 1.43 86.0 ? 0.2 89.3 ? 1.1 0.013 0.59 0.60 Arginine 4.90 3.42 9.48 7.29 79.9 ? 1.1 93.0 ? 0.5 <0.0001 3.92 3.18 Isoleucine 1.98 2.30 3.83 4.90 88.2 ? 0.3 88.5 ? 0.5 0.526 1.75 2.04 Leucine 3.31 3.69 6.40 7.87 89.0 ? 0.2 88.5 ? 0.7 0.341 2.95 3.27 Valine 2.57 2.35 4.97 5.01 87.1 ? 0.2 85.6 ? 1.2 0.153 2.24 2.01 Histidine 0.82 1.18 1.59 2.52 88.7 ? 2.1 89.6 ? 0.7 0.578 0.73 1.06 Phenylalanine 1.96 2.48 3.79 5.29 87.4 ? 0.4 89.8 ? 0.7 0.012 1.71 2.23 Nonessential AA 29.1 25.5 Cystine 0.65 0.63 1.26 1.34 69.4 ? 1.2 79.1 ? 2.3 0.006 0.45 0.50 Serine 2.09 2.52 4.04 5.37 83.0 ? 0.5 87.7 ? 1.0 0.004 1.73 2.21 Proline 1.66 2.14 3.21 4.56 87.9 ? 0.8 87.2 ? 1.2 0.519 1.46 1.87 Alanine 2.80 2.07 5.42 4.41 88.8 ? 0.3 84.7 ? 1.0 0.005 2.49 1.75 Aspartic ac. 4.27 5.49 8.26 11.71 n/d 85.7 ? 0.8 n/d n/d 4.71 Glutamic ac. 13.83 8.81 26.75 18.78 84.8 ? 0.7 90.5 ? 0.6 0.001 11.72 7.97 Glycine 2.34 2.01 4.53 4.29 81.8 ? 0.9 83.3 ? 0.5 0.125 1.92 1.67 Tyrosine 1.48 1.78 2.86 3.80 86.7 ? 0.1 89.4 ? 0.9 0.016 1.28 1.59 n/d: not determined; ?A: microalga; SM: soybean meal; [AA]: amino acid concentration

Example 2.6

Determination of Apparent Metabolizable Energy (AME) and Nitrogen-Corrected Apparent Metabolizable Energy (AMEn) of Strain CC4062/5 Cultivated for 22 h

[0187] The measurement of apparent metabolizable energy (AME) of the microalga microalga, strain 4, 22-h culture was performed in 3-week-old chickens (Bourdillon et al., 1990). The so-called substitution method was applied starting with a control diet composed of corn-soybean base and premix. Like the other raw materials, the microalga, ground on a 3-mm grid, was incorporated in increasing proportions of 4%, 8%, 12%, 16% and 20% in substitution for the corn-soybean mixture, while keeping the premix constant in the diets.

[0188] Table 9 presents the formula of the control corn-soybean feed as well as the specifications thereof (crude protein=18%, apparent metabolizable energy=3200 kcal/kg). The feeds substituted with the microalga are optimally balanced (notably in their protein to energy ratio) on the basis of assumptions regarding AME, digestible amino acids, and bioavailable phosphorus made for the microalga.

TABLE-US-00012 TABLE 9 (test 14ALG069): Formula of the experimental feed supplement used D1 Basal Corn-Soybean Corn 67.673 Soybean meal 48% 19.742 Corn gluten 60 4.766 Soybean oil 3.139 Premix 4.680 Microalga 0 Crude protein 18 Fat 6.201 Crude fiber 3.029 Mineral substances 2.013 Lysine 0.80 Methionine 0.33 Met + Cys 0.664 Threonine 0.686 Tryptophan 0.173 Arginine 1.066 Calcium 0.1 Total phosphorus 0.333 Available phosphorus 0.078 Sodium 0.092 AME, kcal/kg 3200

[0189] Diet 1: basal corn-soybean (95.32%)+vitamin and mineral supplement (VMS) (4.68%)

[0190] Diet 2: basal corn-soybean (91.32%)+VMS (4.68%)+microalga (4%)

[0191] Diet 3: basal corn-soybean (87.32%)+VMS (4.68%)+microalga (8%)

[0192] Diet 4: basal corn-soybean (83.32%)+VMS (4.68%)+microalga (12%)

[0193] Diet 5: basal corn-soybean (79.32%)+VMS (4.68%)+microalga (16%)

[0194] Diet 6: basal corn-soybean (75.32%)+VMS (4.68%)+microalga (20%)

[0195] This incorporation gradient makes it possible to evaluate the AME value of the microalga by simple linear regression. The experimental design is described in FIG. 1. The dry matter values of the diets were determined after grinding and on feed pellets at the beginning and end of the assessment period. Finally, the complete feeds and the feces were analyzed individually for their gross energy (GE) content by calorimetry.

[0196] The AMEn of the feeds is calculated as follows:

[00002]$\mathrm{EMA}?\left(\mathrm{kcal}.Math.\text{/}.Math.\mathrm{kg}.Math..Math.\mathrm{DM}\right)=\frac{\left(I?\mathrm{GEi}-E?\mathrm{GEf}\right)}{I}$$\mathrm{EMAn}?\left(\mathrm{kcal}.Math.\text{/}.Math.\mathrm{kg}.Math..Math.\mathrm{DM}\right)=\mathrm{EMA}-8.22.Math.\frac{\left(\mathrm{WG}?0.18\right)/6.25}{I}$

with I: amount of feed ingested (kg DM); E: amount of excreta (kg); GEi and GEf: gross energy of the feed ingested (kcal/kg DM) and of the feces (kcal/kg); WG: weight gain during the assessment period. The AME values were first nitrogen-corrected on the basis of 18% protein contained in animal tissue and by using the factor 8.22 kcal/kg N (Hill and Anderson, 1958). In parallel, nitrogen-correction calculations were also made based on assay of total nitrogen in the excreta. The two methods giving identical results, it is the latter method which is considered in this report. The outliers (?2.5 standard deviations from the mean) were removed before treatment by analysis of variance (ANOVA, SAS 9.1.3? 2002-2003 by SAS Institute Inc., Cary, N.C., USA) according to a factorial design. Dry matter digestibility (dDM) and the apparent digestibility coefficients of nitrogen (DCa nitrogen), phosphorus (DCa phosphorus), calcium (DCa calcium) and fat (DCa fat) are calculated in the same way for each diet.

[0197] These results were analyzed by linear regression (XLSTAT 2010.4.02? Addinsoft 1995-2010), according to the following model:)

Y.sub.i=?.sub.0+?.sub.jX.sub.ij+?.sub.i

With: Y.sub.i=value observed for the dependent variable for observation i, [0198] ?.sub.0=intercept [0199] ?.sub.j=slope of the regression line [0200] X.sub.ij=value of variable j for observation i (% incorporation of the microalga), [0201] ?.sub.i=model error.

[0202] The value of the parameter for 100% incorporation of the microalga was extrapolated from the prediction model, thus making it possible to estimate AME and AMEn and the apparent digestibility coefficient of phosphorus of the microalga.

[0203] Table 10 presents the dry matter digestibility and nitrogen-corrected apparent metabolizable energy (AMEn) results of the control corn-soybean feed as well as the substituted diets D2 to D6. The apparent digestibility coefficients of nitrogen (DCa nitrogen), phosphorus (DCa phosphorus), calcium (DCa calcium) and fat (DCa fat) were estimated by the same method.

TABLE-US-00013 TABLE 10 (test 14ALG069): Dry matter digestibility, energy digestibility, and apparent digestibility coefficient of the experimental feeds substituted with 0%, 4%, 8%, 12%, 16% and 20% of the microalga tested (strain 4, 22-h culture), respectively, and measured in 23-day-old chickens Experimental feeds Diets D2 D3 D4 D5 D6 D1 BC-S + BC-S + BC-S + BC-S + BC-S + BC-S 4% ?A 8% ?A 12% ?A 16% ?A 20% ?A p-value dDM (%).sup.1 75.10 ? 1.20a 74.41 ? 1.09ab 73.53 ? 1.64b 71.64 ? 1.43c 70.17 ? 1.22d 69.56 ? 2.05d <0.0001 AMEn (kcal/kg DM).sup.2 3398.0 ? 53a 3379.4 ? 42a.sup. 3331.8 ? 64b 3273.6 ? 61c 3218.9 ? 53d 3171.4 ? 72e.sup. <0.0001 GE/AMEn (%).sup.3 78.35 ? 1.34a 78.05 ? 1.25a 77.71 ? 1.72a 76.19 ? 1.56b .sup.75.00 ? 1.56C 74.72 ? 1.84c <0.0001 DCa nitrogen (%).sup.4 61.79 ? 3.75a 60.24 ? 2.44ab 58.80 ? 4.87b 56.48 ? 3.15c 53.97 ? 2.26d 53.90 ? 3.44d <0.0001 DCa phosphorous (%).sup.5 38.00 ? 4.67a 40.78 ? 4.36b 41.42 ? 4.87b 36.96 ? 4.89a 37.77 ? 3.38a 37.28 ? 3.83a 0.004 DCa calcium (%).sup.6 26.84 ? 5.41a 34.32 ? 5.85b 37.86 ? 5.91ab 34.90 ? 4.61bc 36.74 ? 4.55bcd 38.94 ? 5.01d <0.0001 DCa fat (%).sup.7 85.88 ? 4.2ab .sup.87.17 ? 1.88ac 88.59 ? 2.53c 85.93 ? 3.33a 83.97 ? 3.37bd 83.86 ? 3.14d <0.0001 B C-S: basal corn-soybean; ?A: microalga .sup.1Dry matter digestibility (%); .sup.2Nitrogen-corrected apparent metabolizable energy (AMEn); .sup.3Gross energy relative to AMEn (%); .sup.4Apparent digestibility coefficient of nitrogen; .sup.5Apparent digestibility coefficient of phosphorus; .sup.6Apparent digestibility coefficient of calcium, .sup.7Apparent digestibility coefficient of fat (%)

[0204] The dry matter digestibility of the various diets is strongly correlated with their AMEn values. The AMEn of corn-soybean diet D1 is measured at 3398?53 kcal/kg DM. The results show that the AMEn value of diet D2 is equivalent to that of the corn-soybean control and reflect that the 4% substitution with the microalga does not impact the energy digestibility of the diet. In contrast, and starting from an 8% or higher substitution of microalga, AMEn and DCa nitrogen decrease linearly with increasing percentages of substitution (p<0.0001).

[0205] The addition of 4% to 16% microalga shows a reduction in the feed conversion ratio (FCR) over the period 13-23 days. It should also be noted that the chickens tend to consume less (except for D2) for a similar weight gain, however, regardless of the percentage of incorporation of the microalga and higher than that measured for the control corn-soybean diet (Table 11).

TABLE-US-00014 TABLE 11 (test 14ALG069): Weight gain, consumption, and feed conversion ratio of the animals over the period 13 to 23 days fed diets substituted with 0%, 4%, 8%, 12%, 16% and 20% of the microalga tested (strain 4, 22-h culture), respectively. Experimental feeds D2 D3 D4 D5 D6 D1 BC-S + BC-S + BC-S + BC-S + BC-S + BC-S 4% ?A 8% ?A 12% ?A 16% ?A 20% ?A Weight gain (g) 460 ? 58.8 a 496 ? 36.5 b 495 ? 90.0 b.sup. 516 ? 56.6 b 510 ? 37.5 b 493 ? 44.1 ab Consumption (g) 749 ? 82.9 a 756 ? 50.3 a .sup.713 ? 118.8 ab 722 ? 68.0 ab 701 ? 51.9 be 665 ? 52.8 c Feed conversion ratio (g/g) 1.63 ? 0.063 a 1.53 ? 0.048 b 1.44 ? 0.067 c 1.41 ? 0.060 d 1.37 ? 0.043 de 1.35 ? 0.042 e B C-S: basal corn-soybean; ?A: microalga

[0206] The AME and AMEn values of the microalga, strain 4, 22-h culture (Table 12) are measured at 2785 kcal/kg DM and 2296 kcal/kg DM, respectively. It should be noted that these values are in the range of the mean in vivo reference values of the AMEn measurement of soybean meal of protein class 46, 48 and 50 of 2303?137, 2348?248, and 2365?178 kcal/kg DM, respectively. Thus, the energy supply provided by this microalga is comparable to that of standard-quality soybean meal.

TABLE-US-00015 TABLE 12 (test 14ALG069): A: Apparent metabolizable energy and nitrogen-corrected apparent metabolizable energy of the microalga tested measured in 23-day-old chickens. B: In vivo reference values measured according to the same model for various soybean meal protein classes (references from Adisseo, 2012). Micro- Soybean Soybean Soybean alga meal meal meal AME, 2635.0 Protein 46 48 50 kcal/kg class AME, 2785.4 kcal/kg DM AMEn, 2172.0 N n = 15 n = 27 n = 80 kcal/kg AMEn, 2296.0 AMEn, 2303 ? 137 2348 ? 248 2365 ? 178 kcal/kg kcal/kg DM DM A B

Example 2.7

Choice and Consumption Measurement Tests in Chicks of Feeds Comprising the Biomass According to the Invention

[0207] In each test presented below, the microalga, strain 4, 22-h culture, like the other raw materials, was ground on a 3-mm grid and incorporated in increasing proportions of 5%, 10% and 15% into the corn-soybean diet, while adjusting the formulation so as to have four iso-energetic, iso-protein and iso-lysine diets (Table 13), the composition of which is given in Table 14.

TABLE-US-00016 TABLE 13 (test 14ALG069): Apparent digestibility coefficient of phosphorus of the microalga tested measured in 23-day-old chickens by linear regression Microalga Digestible phosphorous (% DM).sup.1 0.86 DCa phosphorous (%).sup.2 35.7 .sup.1Confidence intervals: 0.576%-1.143% .sup.2Calculated according to the formula: DCa = Digestible P (%)/Feed total P (%)

TABLE-US-00017 TABLE 14 (test 14ALG081): Formulas of the experimental feeds used in the consumption and choice tests in chicks 5% 10% 15% Control microalga microalga microalga %, as provided feed feed feed feed Corn 52.90 54.28 55.67 57.06 Soybean meal 48% 35.40 29.50 23.59 17.69 Extruded soybean 4.00 4.00 4.00 4.00 Soybean oil 3.55 3.10 2.65 2.20 Dicalcium phosphate 1.92 1.78 1.63 1.49 Calcium carbonate 1.01 1.13 1.25 1.36 Salt 0.36 0.35 0.34 0.32 DL-Methionine 0.25 0.24 0.23 0.22 L-Lysine HCl 0.01 0.02 0.04 0.06 Premix 0.6 0.6 0.6 0.6 Microalga 0 5 10 15 Crude protein 22 22 22 22 Lysine 1.25 1.25 1.25 1.25 Methionine 0.58 0.59 0.60 0.61 Met + Cys 0.95 0.96 0.96 0.96 Threonine 0.86 0.87 0.88 0.88 Dig Lysine 1.12 1.12 1.12 1.12 Dig Methionine 0.57 0.58 0.58 0.59 Dig Met + Cys 0.85 0.85 0.85 0.85 Dig Threonine 0.74 0.75 0.76 0.77 Dig Tryptophan 0.25 0.24 0.24 0.24 Calcium 0.95 0.95 0.95 0.95 Total phosphorous 0.72 0.77 0.83 0.88 Available 0.40 0.40 0.40 0.40 phosphorous Metabolizable 3000 3000 3000 3000 energy, kcal/kg

[0208] Two experimental tests were carried out in parallel (FIG. 2). In the first test (choice test), consumption is measured when the animal has the choice between a microalga-supplemented feed and a corn-soybean starter. In the second (consumption measurement), consumption is measured when the animal has access to only one feed, optionally supplemented with microalga.

[0209] In test 1, the consumption data at each measurement point at 7 and 9 days of age were analyzed according to a paired test procedure (XLSTAT 2010.4.02? Addinsoft 1995-2010) considering that the consumption from one feeding dish is dependent on the consumption from the other in each cage.

[0210] The consumption and weight data from test 2 were analyzed for a feed effect according to an ANOVA procedure (XLSTAT 2010.4.02? Addinsoft 1995-2010) according to the following model with a 95% confidence interval:

Y.sub.i=?+a.sub.i+b.sub.iY=parameter

[0211] With [0212] Y=parameter [0213] ?=mean [0214] a.sub.i=feed effect [0215] b.sub.i=block effect

[0216] For each significant difference, the means are analyzed by a pairwise Fisher's LSD test.

[0217] 1) Test 1Choice Test

[0218] For the test with choice of feed, approximately 200 1-day-old male chicks (Ross PM3) were placed in groups of about 20 in divided metabolic cages and fed a standard starter containing wheat, corn and soybean meal. At 6 days of age the chickens are starved for 2 hours before being weighed and distributed by weight group. One hundred twenty chickens are thus selected, placed by groups of 4 into 30 divided metabolic cages and assigned at day 7 to one of the experimental treatments according to their weight (10 repetitions per treatment). Each cage contains two feeding dishes containing different feeds, corresponding to the following three experimental treatments: [0219] Treatment 1: corn-soybean starter (feeding dish 1) and 5% microalga (feeding dish 2) [0220] Treatment 2: corn-soybean starter (feeding dish 1) and 10% microalga (feeding dish 2) [0221] Treatment 3: corn-soybean starter (feeding dish 1) and 15% microalga (feeding dish 2)

[0222] Feed and water are distributed ad libitum throughout the test. The experimental feeds are provided as 3.2-mm-diameter pellets. Consumption is measured at T.sub.0+1h, T.sub.0+2h, T.sub.0+3h, T.sub.0+4h and T.sub.0+0h at 7 and 9 days of age, with the feeding dishes in the cages being switched every hour. Between the two consumption measurements (day 8), the animals receive the wheat-corn-soybean starter.

[0223] The results show (see FIGS. 3-5) that the young animals did not reject the microalga-supplemented feeds, and this regardless of the incorporation rate. In these amounts, the microalga thus presents no palatability problem. The young chicks even tend to prefer the feeds supplemented with 5% and 10% microalga (FIG. 5) compared to conventional corn-soybean feed, which is reversed numerically when 15% microalga is added.

[0224] 2) Test 2Consumption Measurement

[0225] In a second test, the chicks have access to only one type of feed, optionally supplemented with microalga (FIG. 6). Approximately 400 1-day-old male chicks (Ross PM3) were weighed and distributed by weight group. Two hundred forty selected animals are placed by groups of 4 at day 1 in 60 undivided cages in blocks of homogeneous weight (15 blocks of 4 cages) with one feeding dish per cage, each with an experimental feed (15 repetitions per feed). In each cage, the 4 chicks are identified individually. Feed and water are distributed ad libitum throughout the test. The experimental feeds are corn-soybean starter supplemented with 0%, 5%, 10% or 15% microalga, also used in test 1 with choice of feed. Individual live weights are measured at 1, 7 and 9 days of age. Uneaten feeds are weighed and consumption per cage is measured at 7 and 9 days of age. From 0 to 7 days of age, the animals fed the feed containing 10% microalga have a significantly improved weight gain relative to the control and to the other two supplemented diets. The feed conversion ratio measured for the 10% supplemented diet is significantly improved relative to the control and to the 15% supplemented diet (1.089 vs 1.156). Furthermore, the supplementation with microalga has no effect on feed consumption when compared with the corn-soybean control (122.6 g, 125.7 g, 135.6 g and 124.6 g, respectively, for the control and the three diets supplemented 5% to 15%).

[0226] Over the total measurement period from 0 to 9 days of age (FIG. 7), the positive effect of the feed supplemented with 10% microalga on weight gain and feed conversion ratio are confirmed.

[0227] The consumption results (under non-choice conditions) show that the microalga, strain 4, 22-h culture tested may be included up to 15% in balanced corn-soybean feeds without affecting the performance of 0- to 9-day-old chicks.

REFERENCES

[0228] Becker E. W. (2007) Biotechnol. Adv., 25: 207-210 Henman et al., 2012 [0229] Bourdillon A., Carr? B., Conan L., Francesch M., Fuentes M., Huyghebaert G., Janssen W. M., Leclercq B., Lessire M., McNab J., Rigoni M. et Wiseman J., 1990b. Br. Poult. Sci., (31), 567-576. [0230] Carr? et Brillouet (1989, Determination of water-insoluble cell walls in feeds:

[0231] interlaboratory study. Journal Association of Official Analytical Chemists, 72,463?467). [0232] Green S., Bertrand S., Duron M., Maillard R., 1987. Br. Poult. Sci., (28), 631-641. [0233] Green S., Bertrand S., Duron M., Maillard R., 1987. Br. Poult. Sci., (28), 631-641. [0234] Henman D. J. (2012) Report 4A-102, CRC Australia, 15 p [0235] Hill F. W. et Anderson D. L., 1958. J. Nutr., (64), 587-603. [0236] Lieve M. L. Laurens, Thomas A. Dempster, Howland D. T. Jones, Edward, J. Wolfrum, Stefanie Van Wychen, Jordan S. P. McAllister, Michelle Rencenberger, Kylea J. Parchert, and Lindsey M. Gloe. Algal Biomass Constituent Analysis: Method Uncertainties and Investigation of the Underlying Measuring Chemistries. Anal. Chem. 2012,84,1879-1887 Bourdillon et al., 1990 [0237] Prosky L., Asp N.-G., Schweitzer T. F., DeVries J. W., Furda I., 1988. Determination of total dietary fiber in foods and food products: interlaboratory study. Journal of the Association of Official Analytical Chemists, 71:1017-1023. [0238] Skrede A., Mydland L. T., Ahlstrom ?., Reitan K. I., Gislerod H. R., ?verland M. (2011) J. Anim. Feed Sci., 20: 131-14211 [0239] Terpstra K., De Hart N., 1974. J. Anim. Physiol. Anim. Nutr., (32), 306-320. [0240] WO 2010/051489 [0241] WO 97/37032