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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
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