mass algal production - reproseed

Advances in algal production

René ROBERT-Workshop Reproseed, 28-29 October 2013, Brest (France)

French team

R. Robert G. Bougaran N. Marc B. PettonL. Lebrun D. Ratiskol

Dutch team

P. Kamermans P. van

Dalen

S.

Merel

A. van

Gool

A.

Quesnot

N. RosC.

Chivard

R.

Maas

• Such promising continuous culture concept has been accordingly proposed at

outdoor production to decrease the cost of phytoplankton biomass production

and thus the running cost of spat.

• The strategy was as followed:

a) Definition of the ecological needs (Temperature/Irradiance/pH) of three diatoms species :

Skeletonema marinoi, Chaetoceros gracilis and Thalassiosira weissflogii.

b) Definition of a low cost medium

c) Feasibility of batch, continuous and semi continuous cultures outdoor using similar paddle raceways

in Argenton-France (3 for 3 diatoms) and in Yerseke-Holland (4 diatoms and 2 flagellates)

d) Use of growth model and weather station data recording to propose efficient rotation of the

cultures year-round.


WorKshop Reproseed, 28-29 October 2013, Brest (France)


Definition of the ecological needs (Temperature/Irradiance/pH) of

three diatoms species : example Chaetoceros gracilis

quadratic model

23* central composite design

Main effects Interactions

17 experiments8 cube angles3 central replicates6 « star points »



17 x 250-mL photobioreactors with

• Temperature control : 10 – 45°C

• pH control (CO2) : 5 – 10

• Dimmable irradiance (LED arrays) : 20 – 1300 µmol.m-2.s-1

The TIP device

pHT°

LEDs

CO2

Heatingdevice

Labview-based supervisor program



Workshop Reproseed, 28-29 October 2013, Brest (France)

Experimental procedures

• Natural sea-water enriched with Si-Walne medium

• Randomized experimental conditions

• Inoculation with optically clear phytoplankton culture

• Sampling (1 mL) twice or three times a day for OD680 and OD800

• Sampling (1 mL) once a day for cell concentration (image analysis)

Temperature (°C) 15 - 35

Irradiance (µmol.m-2.s-1) 20 – 1300

pH 6.0 – 9.0

Factor ranges: defined from literature

• µmax computed from the linear part

of the Ln-transformed growth curve


Worshop Reproseed, 28-29 October 2013, Brest (France)

Correlation between cell concentration and OD680

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7-2

0

2

4

6

8

10

12

14

16

18x 10

6

OD680

Cel

l con

cent

ratio

n (m

L-1)

Chaetoceros gracilis

R = 0.96

µmax can be reliably

computed from the

OD680 data




Growth curves and µmax computation

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 4 6 8

Time (d)

OD

680

c1c2

c3c4

c5c6c7

c8c9

c10c11

c12c13

c14c15c16

c17

Culture µmax (d-1)

C1 0.08

C2 1.80

C3 2.23

C4 2.07

C5 2.25

C6 1.79

C7 0.84

C8 0.67

C9 1.42

C10 1.07

C11 1.97

C12 1.09

C13 2.06

C14 1.67

C15 1.87

C16 0.39

C17 2.17




Factorial design analysis

µm

ax(d

-1)

Irradiance, temperature and pH

have significant positive effects on µmax

(a = 5%)

No significant interactions on µmax (a =

5%)

*

**

Model explains 90% of the variability



Factorial design analysis : optimization

Optimal conditions for

growth rate

I = 930 µmol.m-2.s-1

T = 33 °C

pH = 9.0

µm

ax

(d-1

)µ

ma

x(d

-1)

µm

ax

(d-1

)


Species well adapted for summer conditions



Low-cost medium for Chaetoceros gracilis

After some preliminary tests in 2 ml well plates, four simplified media were

compared to a control (Walne) in 10-l bottles. The highest cell densities and longest

duration of the cultures was obtained with ammonium, phosphorus (N:P ratio 25:1),

silica, iron, manganese and vitamins



Indicator of growth limitation in outdoor cultivation of microalgae

The ratio absorbance at 480 nm

------------------------------

absorbance at 665 nm

may indicate nutrient limitation (ratio above 2)

This was tested with different nutrient treatments

The goal was to predict and avoid the crash of the

culture



Indicator of growth limitation in outdoor cultivation

of Skeletonema marinoï

Nutrient limitation (ratio above 2)

Rapid increase in ratio on day 4 in 0% Walne medium and day 5 in 10% Walne

medium. Ratio works, but predicts too late for farmer to take action

Dilution rate = 20 % d-1

(d = 0.2) for continuous cultures

1 µm filtered seawater

5 L container

plus bubbling

U.V

2m3 raceway 16.6 L h-1Nutrient solution

preparation

3-4 day-old inoculum, varying from 180 to 300L, originated from healthy

cultures cultivated indoor in perspex cylinders.

Peristaltic

pump

UV

lamp





Chaetoceros gracilis continuous culture

Growth of Chaetoceros gracilis, expressed as cell concentration, in

2m3 outdoor batch, continuous (20% daily) and semi-continuous (50%

each second day) conditions (spring 2011).

• Batch lasted 16d

with optimal

concentration

of 2.5 M ¢ ml-1

• Continuous

culture lasted

28d with optimal

concentration of

1.5 million ¢ ml-1

• Semi continuous

lasted 50d with

1.5-1.7 M ¢ ml-1

Argenton



Regardless to the techniques, neither temperature (a) or pH (b) evolution explained the

fluctuations of C. gracilis cultures. From day 48 semi-continuous cultures temperature

reached 22 °C and remained over this value until the end of the experiment (optimum at 24

°C on day 49: a). From day 2 to day 7 pH increased from 8.5 to 9.5-10 in all cultures.

Thereafter batch culture decreased to initial value on day 18 where it collapsed (8.5). For

both other cultures pH remained high between 9-10 until they collapsed (b).

a b Argenton Argenton




Average cell density (n=2 ± sd) of Chaetoceros gracilis continuous culture with Walne

medium and 0.2 μm filtered seawater in raceway with three treatments (control, high

nitrogen, low salinity). Continuous culture with 10% daily renewal rate from day 5

onwards. Collapse of the cultures could not be related to sudden changes in water

temperature, salinity, dissolved oxygen or pH (summer 2011).

Yerseke




Vibrios development often occurred in Skeletonema marinoi whatever the

techniques used (1/2) whereas such contamination was less frequent with the

other diatoms with positive effects of continuous culture on vibrios load decrease



Collecting data from local weather station (Ploudalmézeau)

Last 5 years mean irradiance increased from 50 to 250 W m-2 from January to June and

decreased from June to December in an opposite way with a high monthly variance ; whereas

temperature increased from 7°C in January to 16°C in July and decreased in an asymmetric

manner to 8°C in December.

Such data combined with those achieved from growth model are good indicators for

selecting the type of species to be grown outdoor and to decide species culture rotation

on a year-round. However some cautious has to be taken with the use of temperature



Collecting data from local weather station (Ploudalmézeau)

There is a difference between both types of data that may reach 5-7°C that could be

explained by the recording of temperatures at night in the local weather station and the use

of a lid to limit air born and seawater born contamination. Indeed such translucent lid induce

greenhouse effects.

Air temperature

Culture temp.



1. Chaetoceros gracilis and Thallassiosira weissflogii are both adapted to summer

conditions

2. The ecological requirements of Skeletonema marinoi is unknown today (trials are

in progress) but failures in batch culture when T°C ≥ 22°C indicate a preference

for colder conditions (spring and autumn) during which successful cultures have

been carried out.

3. A simple medium containing ammonium, phosphorus (N:P ratio 25:1), silica,

iron, manganese and vitamins is an appropriate medium for the culture of

diatoms

4. For successful cultures it is necessary to use inoculum that is in good condition

5. C. gracilis is a good candidate for continuous cultures whereas T. weissflogii

seemed to be more adapted for semi-continuous cultures

6. Whereas S. marinoi batch cultures exhibited generally a shorter duration of the

culture, there is no clear overall trend to design the best type of cultures in

outdoor conditions for that species

7. C. gracilis and T. weissflogii exhibited low level of bacteria (total bacteria and

vibrios) especially in continuous or semi-continuous conditions. S. marinoi is the

most easiest contaminated species by bacteria including vibrios.

Main conclusion


That’s all folks!

It is time to leave

Can I give you a lift?


• The production of phytoplankton is a bottleneck for most commercial hatcheries

because the requirements of mollusk is high. This is specifically true for spat stage that

is roughly 100 fold more demanding than larvae: ≈1.5 m3 per million spat < 4 mm vs 15 l

per million larvae (Helm et al 2005).

• Until now most of hatcheries produced phytoplankton by the batch method which is

relatively efficient but labour cost due to frequent cleaning operations.

• Continuous cultures have been commercially developed since the last 10 years and some

hatcheries have adapted this way of producing phytoplankton in indoor conditions

(seaCaps, Biofence, Aqualgae).

• In France in cooperation with ma m jor hatchery managers a new 120L

photobioreactor, modulable and compact have been designed (Olivo,

2007: PhD), improved and tested on t. Iso (pH = 6.8 ± 0.2, Temp = 28

± 1 ° C and irradiance (330 µmol m-2 s-1) at a daily dilution rate of 0.4

d-1 (40%) in a commercial hatche ry (Marchetti 2011: PhD):

production of 27 x 106 cell ml-1 du ring two consecutive months




Only some results have been exposed here due to time restriction but:

LPI (Argenton) has made experiments:

• On batch, continuous and semi continuous cultures in outdoor conditions on Skeletonema

marinoi (22 assays) Chaetoceros gracilis (8 assays) and Thallassiosira weissfloggi (3

assays)

• It also made trials on improvement of the culture on focusing on inoculum (age/initial

volume = concentration + quality of the inoculum), importance of day/night regime on S.

marinoi , influence of the culture techniques on the length of S. marinoi colony), bacterial

evolution throughout all cultures

IMARES (Yerseke) has made assays on:

• Low-cost medium for Pyramimonas parkae

• A test of different treatments of the system to avoid contamination with other algal species

(extra filtration or coverage of raceway)

• Tests of different daily renewal rates (10% or 40%) of continuous culture of Chaetoceros

gracilis and Phaeodactylum tricornutum

• Test of addition of extra inoculum to C. gracilis continuous culture

• Tests of absorption ratio with nutrient and light treatments with Skeletonema marinoi and

Tetraselmis suecica.

mass algal production - reproseed

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