MICROALGAL GROWTH IN THE LAB

AND OUTDOORS

Istituto per lo Studio degli Ecosistemi

LESSON 2- DECEMBER 16, 2014 G. Torzillo

CNR-ISE- ITALY
 Requisite for growth of microalgal biomass culture
are:

1) A viable inoculum

2) An energy source ( LIGHT, Light and organic substrates)

3) Nutrients to provide the essential elements from which the

biomass is synthesized (CO2, macronutrients, micronutrients)

4) Absence of inhibitors which prevent growth

5) Suitable physicochemical conditions (pH, temperature, mixing)

 Important parameters for algal growth evaluation

 (Specific Growth Rate h-1)

 = ln x1 – ln x0 /t

td = ln 2/ = 0.693/

.
 = 0.693/td ( i.e. ln2/td)

Productivity (g/l(h) = µ X V
 EXAMPLE
Xo (biomass concentration) = 3 g/l
X1 (at time t ) = 3.2 g/l

Time (t ), e.g. 1 h
(ln 3,2- ln 3.0 )/1h = 0.0645 h -1
It means that our biomass increases at a rate of 6.4% per
hour. That is, at the start was 3g/l after 1 hour it increased to
3.2 g/l. It is called specific growth rate.

The specific growth rate is analogous to the compound interest rate

on an investiment (in our case 3g/liter biomass invested). Thus, a
specific growth rate of 0,064 h-1, corresponds to a compound interest
rate of 6.4% per hour.

Prod. = µ X V = 0.0645 h-1 x (3.2+3.0/2) g/l= 0.2 g/l/h

 Mode of cultivation

BATCH CULTURE (closed system)

The system is closed if some essential of the system cannot both
enter and leave it. In a batch culture (closed system), with an initial
amount of nutrients, the growth rate of the biomass must tend to
zero, either because of lack of a nutrient or because the further
accumulation of a product can no longer tolerated; hence such
system are always in transient states.

CONTINUOUS CULTURES (open system)

Continuous-flow cultures, on the contrary, have a continuous input of
nutrient medium and output of biomass and other products. At the
steady-state the biomass produced (or compound) will balance the
biomass output.
 DISPOSITIVO DI COLTURA CONTINUA

Motore

Impeller
Sistema di
termostatazione

Sistema di
illiminazione
Reattore
Coltura
overflow
Fotocellula
Mezzo di
Fotometro coltura
Pompa per il
rifornimento di
 =D

The increase in biomass is given by the biomass balance, that is:

(e.g 100 mg) (e.g. 200mg) (100 mg)

Net increase in biomass = growth (productivity) – output
For an infinitely small time interval dt this balance for the whole culture is:

V.dx = V. x.dt – Fx.dt

Dividing throughout by V.dt , and replacing F/V = D we
obtain:
V = culture volume (ml)

dx/dt = ( - D)
In the steady state, when dx/dt = 0, we have

=D
TURBIDOSTAT and CHEMIOSTAT

Chemiostat :
In the chemiostat the dilution rate is fixed and the biomass
concentration will adjust itself to a steady-state level.

D µ
Turbidostat:
The biomass density is fixed and the dilution rate will
adjust itself to the steady-state level

µ D
 CHEMIOSTAT (example)
Cell
Concentration
decreases

D= 0.06 h-1

D= 0.05 h-1 increases
D= 0.04 h-1
PFD = 150 mol photons D = 0.03 h-1
m-2 s-1)
D = 0.02 h-1
D......

Note: if the dilition rate increases above the More and more photolimited. That
maximum growth rate of the culture we reach the is the amount of light received per
washout cell decreases as D decreases.
On the contrary, the cell
Es. D= 0.06 h-1 (6% per hour): concentration increases. For
photosynthetic microorganisms
Volume 1000 ml ; D= 6% = 60 ml/l/h light can be considered as a
substrate. High D allows to cells
(60 ml/l/h)/ 1000 ml = 0.06 h-1 to get more light.
 TURBIDOSTAT

Cell conc= 50 mg/l
Ex. Cell conc= 100 mg/l
PFD = 150 mol photons Cell conc= 200 mg/l
m-2 s-1
Cell conc= 300 mg/l
Cell conc= 400 mg/l
Cell conc= 500 mg/l
Fotoelectric
cell for sensing
light

As soon as the fotoelectric cell gets less

light, fresh medium is added to the
culture until the preceeding status is set.
Set data obtained with a Culture Continue (turbidostat) of Spirulina
platensis (Bocci et al., 1982)

Cell conc Absorbed TD D Prod YEG Biomas

energy produced

(mg/l) (%) (h) (h-1) (g/l/h) (g/Kcal)

Absorbed
energy

35 42 11.0 0.063 2.2 0.007

60 46 11.0 0.063 3.8 0.011

80 55 11.3 0.061 4.8 0.012

100 66 13.0 0.053 5.4 0.012 Better

use of
130 79 15.4 0.045 5.9 0.012 light
200 88 19.2 0.036 7.1 0.012

300 97.5 33.0 0.021 6.4 0.010

510 99.8 63 0.011 5.6 0.008

 Arthrospira platensis (Continuous cultures)

Absorbed Light (%) 100

80

60

40

20
0 100 200 300 400 500 600
Cell conc (mg/l)

Incident light = 3.16 cal m-2 h-1

 Arthrospira platensis (Experiments with continuous culture)

0.10 100
Growth rate

Doubling time (h)

0.08 80
Growth rate (h-1)

TD
0.06 60

0.04 40

0.02 20

0.00 0
0 50 100 150 200 250 300 350 400 450 500 550
Cell concentration (mg/l)l
 P (mg/l/h) = µ (h-1). X (g/l) V (litres)

80 8
Doubling Time (hours)

Productivity

60 6

Productivity (mg/l/h)
40 DT (Doubling time) 4

20 2

0 0
0 100 200 300 400 500 600
Conc. cell (mg/l)
 Relationship between cell concentration and productivity in
Arthrospira platensis (Continue culture experiments)

8 0.07
Productivity (mg/l/h)

0.06

Growth rate (h-1)

Productivity
6 0.05
0.04
4
0.03
2 0.02
Growth rate 0.01
0 0.00
0 100 200 300 400 500
Cell concentration (mg/l)
 Effect of cell concentration on basic culture parameters (Chlorella
sp.) ( Data from Myers & Graham, 1958).
Growth rate (day -1) Efficiency (%).
Concentration (mg/l) Yield (mg/l/day)
400

Efficiency (%); Growth rate (day -1)

5
350
Concentration, Yields

300 4
250
(mg/l)

3
200

150 2
100
1
50

0 0
50 100 150 200 250 300 350 400 450

Mean PFD per cell (mol m-2 s-1)

Effect of biomass concentration on the
biochemical composition of Arthrospira platensis
grown in continuous culture.

100 20
90 Phyc. Protein
80 16

Phycocyanin (%)
70
Protein (%)

60 12
50
40 8
30
20 4
10
0 0
35 60 100 130 200 300 500
Dry weight (mg/l)
Observation: The more dense the culture, the higher the phycocyanin
content. The lower is the growth rate the higher the phycocyanin.
 How environmental factors can affect growth rate and
doubling time.

Growth rate (h-1) TD (hours)

0.07 70

0.06 60

Doubling time (hours)

Growth rate (h-1)

0.05 50

0.04 40

0.03 30

0.02 20

0.01 10

0.00 0
34 36 38 40
Temperature °C

Effect of temperature on growth rate (h-1), and doubling time (h) in

Arthrospira platensis growm continue culture.
 How environmental factors can affect productivity

Productivity (mg/l/h)
10.00
Productivity (mg/L/h)

8.00

6.00

4.00

2.00

0.00
34 36 38 40
Temperature °C
Effect of culture temperature on productivity of Arthrospira
platensis grown in continuos culture.
BATCH CULTURE
Growth measured with Chl Growth measured with dry weight
Calculation of yield of an outdoor culture

 g m-3 day-1 (that is, increase in concentration in a day) * m3 (Volume reactor (m3)

m2 (surface of the reactor)

Yield = g/m2/day

Ex.
g m-3 day-1 = 300 g/m3/ day (increase in biomass in a day)
1 m3 (volume of reactor)
10 m2 (surface of reactor)
300 g/m3 x 0.1 m3/10 m2 = 30 g/m2/day
 Semi-continuous regime (semi-batch).

1.0
0.9
Biomass dry weight (g/l)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
12 24 12 24 12
Time (hours)
 Semi-continuous regime Night loss

Day Night
Dark

Daylight
productivity Net Producivity
 Constant daily dilution (e.g., 40 %)

Biomass dry weight (g/l) 1.4

1.2

1.0

0.8

0.6

0.4
D>u
0.2
A too high dilution rate
0.0
0 1 2 3 4

Time (hours)
 Constant daily dilution (e.g., 10 %)

Biomass dry weight (g/l) 1.4

1.2

1.0

0.8

0.6

0.4

0.2 μ>D
0.0
0 1 2 3 4

Time (days)
 Calculating biomass areal yield in a pond

S/V = 10 m-1 S/V = 20 m-1

Different D.W .
Dry weight = 0.6 g/l Dry weight = 1.2 g/l
Same areal density
Areal density = 60 g/m2 Areal density = 60 g/m2

dept
10-cm 5-cm

100 litres/m2 50 litres/m2

P= 0.2 g/l/d P= 0.4 g/l/d

0.2 g/L x 100 L/m2= 20 0.4 g/L x 50 L/m2 = 20

g/m2/day g/m2/day
 = 1 cm

Cell conc. > 6 g/L

(Dry wt)
S/V = 100 m-1
Areal density :
60 gm-2

Dept = 10-20 cm
Biomass conc. 0.3- 0.6
g/l

S/V = 10 m-1 - 5 m-1

Area density =
60 g /m2
 1.6 cm thick

Florence, Italy (Tredici et al. 1991. Bioresource

Technology
Flat photobioreactors for outdoor culture of microalgae, Israel 1994 (Richmond courtesy)
 Germany (1992)
(O.Pulz courtesy)

+ + +
The Problem of
Light Saturation
 Summary of light satutation irradiances for different
microalgal species reported in the literature.
Species Temperature Saturation irradiances References
(°C) (mol m-2 s-1)

Fresh water Midday PFD = 2000 mol m-2 s-1

Chorella pyrenoidosa 25 115 Phillips & Myers (1954)

Chorella vulgaris 25 80 Sorokin & Krauss (1962)

Chlorella vulgaris 25 125 Aiba (1980)

Chorella pyrenoidosa (2-11-05) 39 225 Sorokin & Krauss (1962)

Scenedesmus obliquus 25 80 Sorokin & Krauss (1962)

Chlamydomonas reinhardtii 25 80 Sorokin & Krauss (1962)

Marine
Diatoms 20 160 Ryther (1956)

Dinoflagellates 20 400 Ryther (1956)

Pheodactylum tricornutum 18 183 Mann & Myers (1968)

Cyanobacteria

Arthrospira platensis 35 170 Bocci et al (1980)

Arthrospira platensis 35 180 Aiba (1979)

Oscillatoria agardthii 20 70 Zevenboom et al. (1980)

Nostoc sp, 30 80 Margheri et al (1991)

 1.0

Photochemistry  PSII
0.8

Fraction of incident light 0.6

irradiation utilised in
photochemistry and heat  NPQ
dissipation 0.4

200 0.2
180  f, D
0.0
160 0 500 1000 1500 2000
-2 -1
140 PFD (mol photons m s )
120
ETR

Light energy not

100 used for
80 photosynthesis
60
Heat dissipation

40
20 Ik
0
0 200 400 600 800 1000 1200

PFD (mol m-2 s -1)

P/I curve (ETR) of Monodus subterraneus cells

recorded through chl fluorescence

measurements.
 The “saturation effect “ of
photosynthesis
1200
2000 1100
s -1 )

1000

1600
-2

900

P ( molO2mg-1Chla h-1)
PFD(  mol photons m

800
700
1200
600
500
800 400
300

400 200
100
0
0 -100
5 7 9 11 13 15 17 19 21 0 400 800 1200 1600 2000
Time of day (hours) PFD ( mol m-2s-1)
Proposed solutions to attenuate the
saturation effect in mass culture

A) Increasing population density

B) Increasing culture turbulence

C) Use special photobioreactor designs

D) Search for strains which have small

antenna size
A) Increasing
population density
 Light gradient
100 PFDin

Photon Flux Density (PFD)

Low biomass
concentration

High biomass
concentration

0
0 L
Depth of culture
Daily time courses of Fv/Fm ratio in Arthrospira platensis (Spirulina) cultures grown
under sunlight in tubular reactors (Czech Rep. Reactor)
0,7

0,6

0,5

0,4
Fv/Fm

0,3
420
730
0,2
1170
2310
0,1

0,0
8 10 12 14 16 18
Daytime
Daily time courses of NPQ in Arthrospira platensis (Spirulina) cultures grown
under sunlight in tubular reactors (Czech Rep. Reactor)

1,0

420
730
1170
0,8 2310

0,6
NPQ

0,4

0,2

0,0
8 10 12 14 16 18
Daytime
Effect of biomass concentration on the daily productivity of Arthrospira
(Spirulina) cultures grown outdoor in photobioreactors.
Tube diameter 5 cm)
600
Gross prod
25 g/m2/day Net prod
Productivity (mg/l/day)

500

400
17.3 /m2/day

300

200

100

0
420 730 1170 2310
Biomass concentration (mg/l)
 Isochrysis galbana
90% shade

Thanks to Hu and Richmond

 Isochrysis galbana
90% shade

60% shade
 90% shade Isochrysis galbana

60% shade

30 % shade
 90% shade
Isochrysis galbana
60% shade;

30% shade

no shade
 90% shade

Isochrysis galbana
60 % shade

30 % shade

No shade
 90% shade

60 % shade

30 % shade

No shade

Arthrospira platensis
Isochrysis galbana
 INCREASING CULTURE MIXING

Mixing of the culture is necessary to:

1) Ensure that all the cells are exposed to light;

2) Maintain nutrient supply throughout the

reactor;

3) Promote oxygen degassing of the culture.

 MOODY CHART
D
a
r
c
y
c
o
e
f
f
i
c
i
e
n
t

f = Darcy coefficient
19 g/m2/day
 Light/Dark ratio

OPTIMIZATION OF LIGHT PATH (Ligh/Dark Cycle)

Tf +Td/ Tf = 5.5 (Best condition); 60 Hz
3 ms+13.5 ms/3 ms = 5.5 ; (1000 ms/ 16.5ms = 60 Hz)
Midpoint
potential
Em7 (Volt)

P700* 3 ps
- 0.9
A0 40-200 ps

A1
15-200 ns
Fx

- 0.6
FA/FB
~ 2µs
P680* 3 ps
Fd
Pheo
200 ps
- 0.3 QA 150-600 s

QB 1 ms
1 ps
0.0
PQ h

f
Cytochrome b6/
Com plex
1-15 ms 1 ms
200 µs
PC
+ 0.3 P700
˜ 1 ms
PSI
H2 0 100-800 s h
+ 0.6 4Mn
200 ns
Yz
O2 P680
+0.9
PSII

Donor side of PSII Acc eptor side of PSII (donor side of PSI) Acceptor side of PSI
 Time cycles for turbulent flow in tubes (Calculation made by dr. Arthur
T. Ippen to assist in exploring the relation of turbulent flow to
intermittent lighting of Chlorella cells). In: J. Burlew (ed.) Algal culture,
from Laboratory to Pilot Plant pag. 261)

50
 10.2 cm
45
 8.89 cm
Tc ( TIME PER CYCLE) IN SECONDS

40  7.62 cm
 6.35 cm
35
 5.08 cm
30
 2.54 cm
25
H= V2/2g + V2 s L/(2g D) + V2 N b/2g
20

15

10 d = 10.2 cm
d = 8.89 cm
d = 7.62 cm
d = 6.35 cm
5 d = 5.08 cm
d = 2.54 cm
0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
V (mean velocity) in m/s
″ The economic realization of a
turbulent flow that would submit
individual cells to a favorable flash
pattern, rather than to a random
distribution of intensity variations,
probably is the major engineering
problem” (Kok, 1953 – Algal
Culture, J.S. Burlew ed. 1953)
 C) Use special photobioreactor designs: light dilution
(Morita et al., 2000, Biotechnol.Bioeng.69: 693-698).

S 2S
A B
A Input light intensity a a

Incident light intensity a a/2

B cos 60° = 0.5
Installation area S S

Photo-receiving area S 2S

Expected prod. X 2X

Results (g/m2/d) 17.1 28.3

Photosynt. Efficiency (%) 4. 1 6. 8

 Light dilution effect

Imax: 1800 μmol photons m-2 s-1 Imax: 400 μmol photons m-2 s-1

(Direct sunlight) (Diluted light effect)

Thanks to Wijffels and Barbosa, 2010 (Science, 329: 796-799)

Light Dilution

1800 400
µmol m-2 s-1
(After Wijffels and Barbosa,

Science 2010)
(Torzillo et al., Biotechnol. Bioeng., 1992
 D
D/2 = 1.57 x D
Patent
pending
M2M Engineering (Italy)
D) Search for strains
which have
small antenna size
PHOTOSYNTHESIS (ETR) VS LIGHT
IRRADIANCE CURVE

ETR = ∆F/F′m x PFD x 0.5 x a*

Chlorella Acclimated to
sorokiniana high light

Acclimated to
low light
 Antenna Size and Photosynthetic Efficiency
Current Wanted

200 Chl 20 20 20 20 20 20 20 20 20 20

Dissipation

Photoinhibition

Photosynthetic Electron-Transport Chain Photosynthetic Electron-Transport Chains

 LIGHT
LIGHT

Absorption

F D

P=  x d
P= yield (g/m2/day) PSII D heat
CORE (photoinhibition)
 = growth rate (day-1)
d = culture depth (m)
P
Electron
Electron Transport
Transport
Chlamydomonas
reinhardtii
 WT

MUTANT

Parameters WT L159I-N230Y
Cell diameter (μm) 7.32 9.43

Chl per cell (μg x 10-6) 4.27 3.32

Productivity (ml H2/Litre) 29 ± 9 504 ± 22

Energy Conversion Efficiency - 2.5 %
Some general principles to reach high productivity

1. Sufficient mixing to expose cell to light and avoid

temperature gradient and biofuling;

2. High mass-transfer capacity to efficiently

supply CO2 and prevent O2 buildup;

3. High surface to volume ratio (S/V) to reach

high cell concentration and volumetric
productivity.
continued

4. Temperature control of the culture close to

the optimum for the grown strain;

5. Accurate control of pH, CO2, and nutrient supply;

6. Adequate harvesting regime to maintain the optimal

concentration;

7. Appropiate choice of the culture system taking into

account the organism that must be cultivated.
FINE
LEZIONE
11/09/2013