Effect of waterborne phytohormones and fish steroids on microalgae Scenedesmus quadricauda

growth and biosynthesis

ABO-2016

I. Algae as an alternative source of:q Energy - biofuelsq Biosynthetic molecules for agri/aquaculture, pharmaceutical and

cosmetic industry

II. Industrial integration (recycling of water, nutrients and energy:q Reduces the cost of productionq Enables to produce variety of co-products

III. Wastewater application:q Hormones expected in aquaponics/hydroponics effluentsq Influence of phytohormones on algae is not well studied

IV. Scenedesmus quadricaudaq High lipids and carotenoids contentq Species of interest for biomass production at Myera Group Inc.

1

} Elucidation of possible influence of aquaponics wastewater components on the metabolic processes in microalgae

} Assessment of influence of waterborne phytohormones: Abscisic acid (ABA), 3-Indoleacetic acid (IAA) and two types of brassinosteroids [brassinolide (BL), 24-epibrassinolide (epiBL)] and fish steroid 17β-estradiol (E2) on growth, biomass production, pigments and lipids biosynthesis of algae S. quadricauda

2

To determine the optimal conditions for enhanced microalgae production for biosynthesis of specific target molecules (neutral lipids and carotenoids)

Ø Batch experiments (aseptically, 7000 lux, 22-23 oC)Ø Growth medium: BBMØ Analyses:

• Water chemistry ions (Gas-Chromatography Mass-spectrometry-GC/MS; DR900 Colorimeter, HACH L. Ltd);

• Algae growth and cells measurement (microscopy-Nikon-Eclipse-Tɩ; NIS-Elements-D3.1software);

• Chlorophyll-a and total carotenoids (spectroscopy BIO-RAD SmaptSpecPlus; concentrations calculated per cellfor methanol solvent);

• Lipids (Gas-Chromatography, Agilent Tech 7890A)• Hormones (ELISA enzyme kit)

3

Hormone Function in vascular plants Algal Concentration

in wastewatersTested range

Degradation in water as ≥50%

Abscisic acid (ABA)

Environmental stresses,

pathogens

Increase: biomass, carotenoids, FAs ≤ 350 µM 1 5-500 µM 6-9 d4

Brassinolide (BL)

Growth, reproduction,

organs differentiation

Increase: biomass, chlorophyll,

monosaccharides≤ 150 nM 2 0.5-300 nM 3-5 d5

24-epi-brassinolide (epi-BL)

As BL,genes damage

Increase: biomass, chlorophyll, proteins ≤ 200 nM2 0.5-100nM 3-5 d5

Auxin (3-Indoleacetic acid, IAA

Growth, tissue differentiation,

metabolism

Increase: biomass, chlorophyll, proteins ≤ 1 mM3

1-1000 nM 5-7 d6

17β-estradiol(E2) Function-fishreproduction

Increase in production, pigments 10-70 nM (NS)

175 nM (S) 5-300 nM 3-8 d 7

References: 1. Kane and Albert, 1989; 2. Nouimli et al 2010; 3. 4. Kane and Albert, 1989; 5. Best et al, 2014; 6. Nissen and Sutter, 1990; 7. Jobling and Owen, 2013. 4

N=Xi –(Xs-Xc), where N-final cell density, Xi – cell density in a hormone-solvent trial, Xs –cell density in a solvent only trial, Xc- cell density in control.

ABA Cell size, µm

Biomass, g/L

pH [initial 7±0.1]

control 45.36 ±6.14 0.46 ±0.04 9.49 ±0.03

5µM 46.77 ±5.26 0.77 ±0.04* 9.63 ±0.17

10µM 43.65 ±4.73 0.58 ±0.03* 9.34 ±0.14

50µM 35.12 ±2.31* 0.59 ±0.05* 9.32 ±0.12

100µM 32.45 ±3.07* 0.37 ±0.02* 9.30 ±0.05

500µM 28.34 ±2.44* 0.33 ±0.04* 8.90 ±0.06

5

0255075

100125150175200

0 3 6 8 11

cells,x10

4 /ml

days

ABA+DMSONa=(Nt –N0)/t,Gotelli,2008

0

50

100

150

200

0 3 6 8 11

cells,x10

4 /ml

days

DMSO

0

50

100

150

200

0 3 6 8 11

cells,x104/ml

days

ABA-DMSOwithcontrolcorrection

control5µM10µM50µM100µM500µM

Chlorophyll-a per cell

0

20

40

60

80

100

120

3 6 8 11

Totalcaroten

,ng/cell

Days

* * ** * *

0

20

40

60

80

100

120

3 6 8 11

Chlor-a

,ng/cell

Days

control5µM10µM50µM100µM

* ***

**

* significant difference from the control (P<0.05). Red asterisks * indicate significant reduction of a parameter.

Total carotenoids per cell

Fatty acids distribution

*

* *

**

**

*

*

*

*

6

0

10

20

30

40

50

control 5µM 10µM 50µM 100µM

FA.m

g/goil

C12:0 C16:0 C16:1C18:1n9c C20:0 C18:3n3

*

*

*

*

*

*

*

*** *

*

*

*

Brassinolide-1 (BL)

* *

Brassinosteroids: S. quadricauda growth

0

50

100

150

200

250

300

0 2 4 6 11 15

cells,x10

4 /ml

days

Epi-BLcontrol0.5nM2nM10nM100nM

BL control 0.5 nM 5nM 50nM 100 nM 300 nM

Cell size, µm 41.96 ±1.75 45.72 ±1.07 53.75 ±1.02* 38.44 ±0.66* 38.82 ±0.49* 36.02 ±0.75*Biomass, g/L 0.44 ±0.03 0.48 ±0.05 0.61 ±0.06* 0.75 ±0.08* 0.59 ±0.05* 0.50 ±0.07

pH [7±0.1initial] 9.02 ±0.03 9.08 ±0.06 8.95 ±0.07 8.89 ±0.1 9.12 ±0.11 8.92 ±0.05

Epi -BL control 0.5nM 2nM 10nM 100nM

Cell size, µm 43.16 ±0.81 50.82 ±2.31* 58.14 ±0.35* 45.05 ±1.05* 38.82 ±0.49*

Biomass, g/L 0.47 ±0.03 0.60 ±0.06* 0.97 ±0.11* 1.17 ±0.09* 0.69 ±0.05*

pH [7±0.1initial] 9.34 ±0.03 9.25 ±0.05 9.25 ±0.08 9.32 ±0.07 9.12 ±0.11

0

50

100

150

200

250

300

0 2 4 6 10 15 20

cells,x104/ml

days

BLcontrol0.5nM5nM50nM100nM300nM

7

Brassinolide-1 (BL)

0

50

100

150

200

250

2 4 6 15

Chlo

r-a,

ng/

cell

days

controlEB 0.5nMEB 2nMEB 10nMEB 100nM

**

*

**

* * *

Chlorophyll-a per cell

24-epibrassinolide(epiBL)

0

50

100

150

200

250

2 4 6 15

Tota

l car

, ng/

cell

days

**

*

* * **

*

**

Brassinosteroids: S. quadricauda biosynthesis

Total carotenoids per cell

*

Brassinolide

***

**

0

20

40

60

80

100

120

140

2 4 6 10 15 20

Chlor-a

,ng/cell

days

**

*

*

*

*

**

*

** *****

**

***

**

Fatty Acids

0

20

40

60

80

100

120

140

2 4 6 10 15 20Totalcaroten

,ng/cell

days

control0.5nM5nM50nM100nM300nM

*** **

*

8

0

10

20

30

contr 5nM 50nM 100nM 300nM

FA,m

g/goil

C16:0 C16:1 C18:0C18:1n9c C18:2n6t C18:3n6

*

**

*

* **

*

**

*

*

0

10

20

30

40

0 0.5 2 10 50 100FA

.mg/goil

EB,nM

C12:0 C16:0 C16:1C18:0 C18:1n9c C18:3n6C21:0

Algae growth in IAA trial

Chlorophyll-a per cell Total carotenoids per cell

0

50

100

150

200

2 4 6 15

Tota

l car

ot,

ng/c

ell

days

* ** *

*

* ** *

*

3-Indoleacetic acid (IAA): S. quadricauda growth and biosynthesis

BL Cell size, µm Biomass, g/L pH [7±0.1 initial]

control 43.72 ±3.75 0.47 ±0.05 8.71 ±0.081 nM 44.24 ±1.33 0.60 ±0.06* 8.81 ±0.085 nM 49.02 ±3.43* 0.70 ±0.07* 8.89 ±0.10

10 nM 49.85 ±2.11* 0.80 ±0.05* 8.85 ±0.09

50 nM 48.05 ±2.31 0.76 ±0.05* 8.65 ±0.11

100 nM 45.44 ±2.16 0.70 ±0.04* 8.82 ±0.04

1 µM 44.12 ±1.48 0.68 ±0.05* 8.62 ±0.10

Fatty Acids

0

50

100

150

200

250

300

350

0 2 4 6 10 15 20

cells,x10

4 /ml

days

control1nM5nM10nM50nM100nM1µM

0

50

100

150

200

2 4 6 15

Chlor-a

,ng/cell

days

control 1nM5nM 10nM50nM 100nM1µM

*

*** *

*

*

*

**

*

*

9

0

10

20

30

40

0 5 nM 10 nM 50 nM 100 nM

1 uMFA

, mg/

g oi

lIAA concentr

C12:0 C16:0 C16:1C18:0 C18:1n9c C20:0C18:3n6 C21:0

*

* * *

*

** *

**

Waterborne 17β-estradiol (E2)

050

100150200250300350400

0 1 2 3 4 5 6 7 8 9 10 11

cells

, x10

4 /ml

Days

control15 ng/l50 ng/l100 ng/l300 ng/l

E2-second set

0

50

100

150

200

250

300

350

0 2 4 6 10 15 18

cell,x104/ml

Days

control

5ng/l

15ng/l

50ng/l

0

20

40

60

80

100

120

140

160

2 4 6 10 15

Totalcaroten

oids,n

g/cell

Days

0

5ng/l

15ng/l

50ng/l*

***

*

*****

*

*

****

**

0

20

40

60

80

100

120

140

2 4 6 10 15 20

Chlor-a

,ng/cell

days

*

*

*

*

*

*

**

*

** ****

***

***

**

I. All tested phytohormones stimulated S. quadricaudagrowth & biomass production

II. All tested phytohormones stimulated algae biosynthesis of pigments and fatty acids

III. Both, biomass production and biosynthesis of valuable molecules can be enhanced by the tested phytohormones

IV. The greatest effect on algae growth and biosynthesis in this study gave IAA and E2 hormones

V. Some key FAs biosynthesis was stimulated by all the tested hormones

VI. Toxic effect of high tested concentrations was observed in the assessment of biomass (ABA), cell size (ABA, BL, epi-BL), growth (BL), pigments (BL) and lipids (ABA, BL)

12

10

0255075

100125150175200225250

2 4 6 11 15

cells

, x10

4 /ml

day

EB 0.5nM +IAAEB 0.5nMEB 0.5+IAA 5nMIAA 5 nMEB 0.5+IAA 100nMIAA 100 nMEB 0.5+IAA 1000nM *

**

*

*

0255075

100125150175200225250

2 4 6 11 15days

EB 2nM +IAAEB 2EB 2+IAA 5nMIAA 5nMEB 2+IAA 100nMIAA 100nM

*

*

*

*

0255075

100125150175200225

2 4 6 15days

Chlor-a, EB 0.5nM EB 0.5nME B 0.5+IAA5nMIAA 5nMEB 0.5+IAA100nMIAA 100nMEB 0.5+IAA1uMIAA 1uM

*

*

* * * *

0255075

100125150175200225

2 4 6 15Days

Chlor-a, EB 2nM EB 2nME 2+IAA5nMIAA 5nMEB 2+IAA100nMIAA 100nM

*

*

**

***

} BioSystems Engineering Department, UofManitoba, Dr. David Levin lab

} Myera Group Inc. – a new company: algae biomass/bioproducts, Winnipeg, MB, Canada

} NSERC, Mitecs

13

Questions?