Seaweeds & Microalgae 3/9/2012

1

Laboratory of Algal Biotechnology Institute of Microbiology Třeboń

Microalgae Biomass as Fish Feed Supplement Jiří MASOJÍDEK Institute of Microbiology, Academy of Sciences, Třeboň Faculty of Science, University of South Bohemia, České Budějovice

Czech Republic Seaweeds & Microalgae 3/9/2012

2 Aqua 2012, Prague

Acknowledgement to co-workers Experimental work

M. Sergejevová, S. Pekařová, J. Kopecký, P. Souček, L. Samcová, P. Novotný, Institute of Microbiology, Academy of Sciences, Třeboň P. Kozák, A. Kouba, J. Velíšek, A. Stará Research Institute of Fish Culture and Hydrobiology, Faculty of Fisheries, Vodňany

Funding

- Czech Science Foundation – project 521/09/0656 - Ministry of Education - ALGATECH CZ.1.05/2.1.00/03.0110,

CENAKVA CZ.1.05/2.1.00/01.0024

- Czech Academy of Sciences

Seaweeds & Microalgae 3/9/2012

3 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Outline • Microalgae biomass as feed supplement • Cultivation of Chlorella – cascade units in Třeboň.

• Case studies - fish feed with Chlorella as antioxidative ‘agent’

Seaweeds & Microalgae 3/9/2012

4 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Microalgae biomass Microalgae aquaculture de novo synthesis • Carotenoids • PUFAs • Accumulation of elements (Se, Cr, I) • Proteins • Pigments • Lipids • Polysacharides • Essential aminoacids • Vitamins

Fish aquaculture Microalgae in feed Positively effect: • Health • Physical condition • Colour • Antioxidative systems • Scavenging of reactive radicals

Seaweeds & Microalgae 3/9/2012

5 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Freshwater unicellular microalga Chlorella Strains of Chlorella vulgaris

• Fast growing microalgae, homogenous, small globular cells • Wide range of temperatures 20-36°C • Phototrophic or heterotrophic cultivation • Source of biomass rich in carotenoids - lutein, violaxanthin, neoxanthin, βcaroten - as antioxidants • Can be enriched by various elements from media - Se

Seaweeds & Microalgae 3/9/2012

10 µm

6 Aqua 2012, Prague

Cascades – unique & excellent production system for microalgae cultivation, designed in the 1960s High productivity (per area & per volume) Microalgae grow in thin layer (~ 6 mm) on smooth declined surface Surface/volume ratio >100 m-1

Masojídek J, Kopecký J, Giannelli L, Torzillo G (2010) Productivity correlated to photobiochemical performance of Chlorella mass cultures grown outdoors in thinlayer cascades. Journal of Industrial Microbiology & Biotechnology 38, 307-317 7 Seaweeds & Microalgae 3/9/2012

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Cultivation of Chlorella in thin- layer cascades • Natural irradiance - max. 2.1 mmol PAR m-2s-1 • High average irradiance per cell, highly turbulent system • High quality biomass - pigment content • Biomass density: 2-40 g L-1 • Manipulation of cultivation conditions to modify biomass quality • Cultivation regime: fed-batch, semi-continuous • About 1.5 tons of high-quality biomass produced per year • Mostly used as human and animal nutrition supplement Seaweeds & Microalgae 3/9/2012

8 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Schematic diagram of mass production of microalgae in aquaculture

Seaweeds Farmers & Microalgae Day, 4/9/2012 3/9/2012

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Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Experimental – fish aquaculture trials

Feed • spray-dried Chlorella biomass is added to feed, 3 to 4-mm sized pellets Freshwater fish species • fingerlings (1 year) - weight 15-25 g, body length 12-18 cm • Experiment • indoor tanks or aquaria, triplicates (12 individuals in each). • 10 days acclimation on basal feed • 6 week on diet • Analysis • pigments – solvent extraction, spectrometry and HPLC analysis • Enzyme activities • Selenium – ICP-MS Seaweeds & Microalgae 3/9/2012

10 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Case studies Microalgae Biomass against Oxidative Stress 1. Barbel (Barbus barbus) & organic and inorganic Selenium in Feed 2. Common carp (Cyprinus carpio carpio) & Carotenoid-rich Chlorella biomass

Seaweeds & Microalgae 3/9/2012

11 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Aim of experiments

Case study 1

Se-enriched microalgae biomass (organic form) and inorganic salts of selenium Na2SeO3 were compared as fish feed supplement to prevent oxidative stress. Experimental Diets Se 0.3 (inorganic form Na2SeO3) 0.3 mg Se/kg Algae 0.3 (organic form Se in algae) 0.3 mg Se/kg Algae 0.9 (organic form Se in algae) 0.9 mg Se/kg Control Seaweeds & Microalgae 3/9/2012

12 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

? Why Selenium ? • Selenium - a co-factor of glutathion peroxidase which active site contains Se-cystein. GPx activity significantly reduced under Se-deficiency. • Glutathion - a key role in the cell in detoxification of reactive radicals by reacting with H2O2 and organic peroxides, the harmful by-products of aerobic life. • Demand for Se – narrow range. Se-cystein • Under some circumstances, the food supplementation is required - Se Se sources added to fish diets • Inorganic form as Na2SeO3 or Na2SeO4 can be toxic. • Organically-bound Se is preferred. Seaweeds & Microalgae 3/9/2012

13 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Selenium accumulation Selenium Selenium in in muscle muscle

Selenium in liver 1.0

0.8 0.8 0.6 0.6 0.4 0.4

aa

ab ab

bb

cc

0.2 0.2 0.0 0.0

Control Control Se Se 0.3 0.3

Algae Algae 0.3 0.3

Algae Algae 0.9 0.9

(mg.kg-1 DW)

-1 DW) (mg.kg-1

1.0 1.0

0.8 0.6 0.4

a

0.2

ab

bc

c

Algae 0.3

Algae 0.9

0.0

Control Se 0.3

ab c bc a Fish accumulated Selenium more readily from Algadiet (0.3 mg Se kg-1 feed) diet as compared to Na2SeO3 (0.3 mg Se kg-1 feed) in muscle and mainly in liver. Seaweeds & Microalgae 3/9/2012

14 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Antioxidant enzyme activity

(µkat L-1)

4 3 2 1

a

b

ab

c

0

Control Se 0.3

Algae 0.3

Algae 0.9

Creatine kinase (CK) 50

(µkat L-1)

5

Alanine aminotransferase (ALT)

40 30 20 10

a

b

b

a

0 Control Se 0.3 Algae 0.3

Algae 0.9

When inorganic form (Na2SeO3 - 0.3 mg Se kg-1 feed) was applied (similarly as in Algae 0.9 mg Se kg-1 feed), Alanine aminotransferase and Creatin kinase activity in blood plasma increased, suggesting stress-induced activity induction in liver … Seaweeds & Microalgae 3/9/2012

15 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Antioxidant enzyme activity 6 4 2 0

a

b

c

a

Control Se 0.3 Algae 0.3

Algae 0.9

(mU.g protein-1)

(mU.g protein-1)

Catalase in muscle

Catalase in liver

60 40 20

a

bc

c

b

0 Control Se 0.3 Algae 0.3

Algae 0.9

… and Catalase activity corresponding to oxidative stress tends to be elevated → indication of Se-overdose. Seaweeds & Microalgae 3/9/2012

16 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Conclusions • Se-enriched microalgae biomass is an effective, safe and promising feed supplement in fish aquaculture. • … Organically-bound Se is more effective in depositing and less toxic than inorganic forms. • The demand for Se is strictly limited and can be easily overdosed. MS in preparation: Antonin Kouba, Josef Velíšek, Alžbeta Stará, Pavel Kozák, Jiří Masojídek: Organic and inorganic forms of Selenium supplied as feed supplement to Barbel (Barbus barbus L.): Its accumulation and biochemical effects.

17 Aquaculture Europe 2011, Rhodes

Case study 2 The resistence of common carp juveniles to oxidative stress (poster No. 528 by M. Sergejevova et al.)

18 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Aim of experiments: Carotenoid-rich Chlorella biomass was tested as fish feed supplement to prevent oxidative stress caused by Chloramine treatment. • Simulation of oxidative stress by Chloramine T (N-deprotonated sulfonamide) − used as biocide and disinfectant to treat various diseases in fish aquaculture • degradation to -ClO-, (hypochroride) production of ROS – oxidative stress. • contraindication - mobilisation of enzyme system

Seaweeds & Microalgae 3/9/2012

19 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Experimental • Fingerlings of common carp (Cyprinus carpio carpio), experiments in triplicates, 12 fish each • 10-day acclimation of fish (fed on basal diet) followed by 6-week feeding on Algadiets with various addition of 1, 2, 5 and 10% Chlorella biomass containing 0.3-0.4% carotenoids in biomass. • Then, fish were washed 3-times in Chloramine T (3 x 1 h, 10 mg L-1) • Final growth, survival, carotenoid content and activity of selected enzymes were evaluated.

20 Aqua 2012, Prague

Colour change of head skin influence by Chlorella supplement in feed of common carp 6-week experiment, juvenile fish, 10 g Muscle - carotenoid accumulation – changes in colour after 1 week – abdomen part, gill cover 0

1

2

5

10 % Chlorella

control Control 0% Chlorella

Algadiet 10% Chlorella

21 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Carp - Carotenoid content in feed and flesh Algadiets - 1-10% of Chlorella in fish feed

250

2.14

200

213.6 1.62 1.52

1.5

150

1.24

1.0

100

105.3

0.5

0.48

50

Carotenods in feed [mg/kg]

Carotenoids in flesh (mg/kg)

2.0

42.8

0.0

8.6

D1 (0%)

21.0

0 D2Seaweeds (1%) & Microalgae D3 (2%)3/9/2012 D4 (5%)

D5 (10%)

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Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

RESULTS Fast growth in all experimental groups - SGR 1.9–2.1 % d-1 no statistical differences between treatments. • After exposure to Chloramine-T, fish groups fed on Algadiets with lower supplement of Chlorella – decrease of antioxidative enzyme activities under physiological values. • The enzyme activity in groups fed on Algadiet with 5 and 10% Chlorella after exposure to chloramine-T were within physiological values of untreated fish.

Seaweeds & Microalgae 3/9/2012

23 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

60 40

GR [mU.mg protein-1]

20

60

40

20

SOD [mU.mg protein-1]

CAT [mU.mg protein-1]

30

GPx [mU.mg protein-1]

Selected enzymes indicating antioxidative activity (lipid peroxidase, superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase) were monitored in target tissues - muscle and hepatopancreas.

80

20

10

Haepatopancreas

1200 1000

GR [mU.mg protein-1]

GPx [mU.mg protein-1]

Flesh Muscle

Antioxidative enzyme activity after Chloramine treatment

800 600

80

60

40

500

400

300

200

Basal

AD1

AD2

AD5 AD10

Seaweeds & Microalgae 3/9/2012

Basal

AD1

AD2

AD5 AD10

24 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Conclusions - In fish groups fed on Algadiets with the decreasing amount of Chlorella - increased symptoms of oxidative stress which led to the exhaustion of antioxidative system capacity. - The enzyme activity in fish groups fed on Algadiet with 5 and 10% Chlorella – no symptom of oxidative stress after Chloramine T treatment - the enzyme activities were within physiological values of the control group (untreated fish). - The lowest antioxidative enzyme activity in the control group fed on normal diet without microalgae. - These results point to the positive influence of the microalgae biomass and its biologically active substances, e.g. antioxidants, such as carotenoids.

Seaweeds & Microalgae 3/9/2012

25 Aqua 2012, Prague

Thank you for attention … Questions? Jiří Masojídek Institute of Microbiology, Academy of Science, Třeboň Institute of Physical Biology, University of South Bohemia, Nové Hrady 26 Aquaculture Europe 2011, Rhodes

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Seaweeds & Microalgae 3/9/2012

27 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Distribution of carotenoids Distribution of carotenoids in Fish meat in Algadiets Pigments Neoxanthin Violaxanthin Anteraxanthin

% 2.4 5.6 0.6

Lutein cis-Lutein X beta-Carotene

64 2.3 6.5 16.8

Pigments Neoxanthin Violaxanthin Anteraxanthin X Lutein cis-Lutein X beta-Carotene

Seaweeds & Microalgae 3/9/2012

% 0 0 6-7 5-9 50-70 4-10 4 -7 0 28 Aqua 2012, Prague

Laboratory of Algal Biotechnology Institute of Microbiology Třeboň, Czech Republic

Optimisation of cultivation process Monitoring chlorophyll fluorescence in-

situ/on-line

Fluorimeter PAM 101-103 (H.Walz)

Seaweeds & Microalgae 3/9/2012

29 Aqua 2012, Prague