IMPAQ - IMProvement of AQuaculture high quality fish fry production.

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IMPAQ Indoor RAS manual

Page
1


IMPAQ
-

IMProvement of AQuaculture high quality fish fry production.

How to in
tensify the production of

copepods as live prey:














By Per M. Jepsen & Benni W. Hansen


DYNAMIC
Status report April 2013



IMPAQ Indoor RAS manual

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2


Background:

IMPAQ is a multi
-
disciplinary Research Alliance that aims at developing a sustainable live
feed in terms of copepods, to supply Danish and International aquaculture fish hatcheries
with a feed item that can be used t
o produce high value fish larvae. The projects below will
focus on intensive copepod production for cold storage of eggs to supply live feed for marine
fish larvae world
-
wide. To supply a stable and reliable flow of cold stored eggs for marine fish
farms,
it is important for the copepod production to establish the entire value
.

Further it is
important to describe the production cost, and compare it with current live feed market to
validate the economic feasibility.

For an intensive copepod egg production

the value chain can be divided into
three

main links.

1.

Algae production as food supply for the copepod culture

2.

Copepod rearing facility

with high densities and

egg

production


3.

Economic evaluation of
the production

Algae production

The algae production facility is located at Roskilde University. It is system based on input
MINH/BENN
i

Copepod production

Copepod cultures can be divided into intensive and extensive systems


the latter are uncontrollable
and thus neglected here. Within
intensive calanoid copepod systems, there are numerous
descriptions of laboratory cultures (< 100L).

To our knowledge only a few intensive systems a

Dutch system, Sintef in Norway and
a newly established

system at RUC. The system at RUC is
based on Recirc
ulated Aquaculture System [RAS] with technology and experience from the Danish
aquaculture sector (13) (16). Two other
intensive
systems

exist, in Australia
and Italy, but are not in
operation anymore.

The copepod rearing facility is at RUC is an unique sy
stem designed in
cooperation between a Danish RAS supplier and RUCs scientific experience with rearing of
copepods.

Economic Feasibility of Intensive Copepod Production for Commercial Scale: A Lab
Experiment

It is well
-
documented in the literature that us
ing Copepods as a live
-
feed has numerous advantages
over the commonly used live
-
feed (Artemia and rotifers). For example, Copepods have a better
nutritional quality, increase survival rate, improve growth condition, reduce mal
-
pigmentation,
enhance develop
ment of key organs, rise success of restocking programs and allows breeding of
new species (Guillaume Drillet). However, the paradox is that they are not widely used and they
remain less available in the market. One of the reasons is economic feasibility a
nd lack of
awareness. In this research, we are going to explore the economic feasibility of intensive copepod
production for commercial scale. Data from Roskilde University lab experiment are used. Cost
benefit analysis is employed (without incorporating t
he monetized values of benefits of copepod on
the entire food chain). We assumed that the benefits of the copepods on the entire food chain are
IMPAQ Indoor RAS manual

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3


reflected on their price. The result reveals that intensive copepod production for commercial scale
can actually

payoff.

1.

Aim of Algae production

The objective is to rear algae in a novel Photo Bio Reactor (PBR).
The chosen alga is
Rhodomonas
salina

since it is proven to be the best feed for the calanoids copepods (Buttino et al. 2012). The
algae part is divide
d

into two parts, one focusing on production and optimization of algae and
another on the link between algae and copepods, the feeding of algae to copepods.

Algae production and optimization:
The optimal
l
ight conditions for
algae
culture

will be
determined
. The optimum is

to prevent

photo

inhibition (at low
alga
concentration) and light
limitation (at high

alga

concentration)
. This is found by adjusting the culture light at different
intensities.

The optimal

climate
for
culture conditions

will be determined. PH will be measured and adjusted
by
the addition of CO
2

(sufficiently)
.

Determine the biochemical content of alga.
To ensure that the
algae have

a high nutritional quality
for the copepods following parameters will be determined in the algae:




Carbon content (CNA analyzer)



Nitrogen content (CNA analyzer)



Fatty acids (GC/MS)



Amino acids (HPLC)



Chlorophyll a content (Spectrophometry)

Determination o
f optimal feeding regime for copepods.
Experiments will determine the
sedimentation of algae in culture systems, and the optimal feeding strategy for copepods.

Approach of the project

Two

PBRs has been developed for intensive cultivation of
R. salina
. The PBR is equipped with
sensors and controlled by Programmable Logic Control (PLC) is used since it is a labour efficient
system. Different challenged in the project will be solved with everything from fully controlled
laboratory setups to big scale exp
eriments in the total PBR included with interactions with the RAS.

The goal is to effectively utilize as much algae for copepod food as possible, with less possible
labour effort.


Algae cultures

Algae cultures are essential as food for copepods.
Hi
gh quality algae have

to be available for the
copepods since the copepods fitness is highly dependent on the quality of food.

The
algae used in
IMPAQ are

Rhodomonas salina
, which in numerous studies has shown to be optimal for copepods
in terms
of
nutritio
nal value and size (
Berggreen et al. 1988;
Hansen 1991).

In the IMPAQ project
IMPAQ Indoor RAS manual

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4


three different methods are used to supply copepods with food. 1. Algae in bottles (Extensive), 2.
Algae in bags (Semi
-
intensive)
,

and 3.
An

algae bioreactor (Intensive).

Ext
ensive (bottles)

Extensi
ve algae are reared in round

bottomed bottles,
which together with atmospheric air help

keeping algae in
suspension. Air supply is filtered with a GF/F 20mm
syringe filter before supplied to the algae with a
10mL
glass pipette. The algae are placed in front of LCD light
panels that supply light that enhances algae growth (LCD
Light Tubes). The light system can be adjusted to three
different light intensities for flexibility so other algae can
be grown.
Every w
eek a backup of the algae are stored
and kept for eventually culture crash situations.
The
volume varies from 2 to 6L depend on the bottle size.
This gives a daily harvest of maximum 2L pr. bottle.



Semi
-
in
tensive (plastic bags)

Semi
-
intensive algae are
cultivat
ed in plastic bags for larger amount
of algae. The system is the same as for bottles except that the
production is in bags. In IMPAQ bags of 15L are routinely used for
up scaling of algae production. This gives a daily harv
est of at least
5L pr. bag.
The algae are kept in suspension with air bubbles, but
often more sedimentation of algae are experienced in bags
compared to bottles.
Bags have

the advantage that algae can be
cultivated for up till 1 month in the bag, and then
the bag can be
discharged. Also if a bag crashes the bag can easily be emptied and
trashed. With bottles or bioreactors
these has to be
thoroughly

cleaned before they can be reused. A bag is trashed and a new bag is installed, facilitating easy
management.






Figure
2

is
a picture of a 15L algae bag installed in the IM
PAQ
project. The reared algae are

Rhodomonas salina
.

Figure
1

is
Rhodomonas
salina

reared in both round
bottomed bottles and in blue cap bottles.

IMPAQ Indoor RAS manual

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5


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Intensive (Bioreactors)


Figure
3

A
conceptual flow diagram of the two algae bioreactor
s

installed
at RUC
-
ENSPAC
for use in

the IMPAQ project.

The system consist of two individual sub
-
units, each sub
-
unit are completely differentiated from the
other. The system is integrated into the existing copepod RAS.

Water supply

(1)
:

To avoid
contamination of the algae cultures
the water quality must be at as high
standard as possible.
The s
eawater
used for
the algae bioreactors
should
be
kept
free of organisms which
compete with the algae.
Competitive
o
rganisms include other types of
phytoplankton, phytophagous
zooplankton and bacteria. Therefore,
the installed
water supply
is filtered
through
0.
2µm
Millipore
finefiltration

syste
m,
after

the
sequential filters an UV system
is


I


2


3


4


5


6


7

IMPAQ Indoor RAS manual

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6


installed, delivering a dose of 240,000mv/cm² at a flow of 1
.5
L/h to ensure clean water for algal
growth.

Air supply

(2)
:

The second most likely source of contamination in algal cultures is the air supply.
M
icro
-
organisms

can multiply quickly within the supply system, particularly if it has condensation.
All air supplies into the algae room
are

filtered
with a

0.2µm
WHATMAN filter
and
are

oil free.
The definition of
a
ir is atmospheric air, and not purified ox
ygen.

Temperature

(3)
:

Temperatures
are

regulated
, since

l
ow temperatures will reduce algal growth
rates and therefore production, whilst higher temperatures will lead to an increased likelihood of
crashes and contamination within the system. The upper and lower thermal limits will be different
for different alga
l strains, and the systems sub
-
units
are therefore

flexible so more than one algal
strain can be produced simultaneously. Temperature changes should be gradual in terms of both air
and water to avoid the risk of shocking the algae. Additions of water shoul
d be of the same
temperature as that of the culture volume.

The water is cooled t
h
rough Roskilde Universit
y’s

central
cooling system, and is externally supplied to the algae bioreactors with air, to prevent contact and a
source for contamination.


CO
2

and
pH

(4)
:

The system
has an integrated
Proportional

Integral

Derivative (PID) controlled
solenoid valve that doses CO
2

into the algae cultures depending on the pH inside the individual
culture. The pH probe and PID control
are

connected and controlled by

a P
rogrammable Logic

C
ontrol system

(PLC)
.

Algae collection and feeding (5):

From the top of each algae bioreactor
the produced algae will
flow into
an

algae collection tank. From this tank the algae can be pumped with four individual
pumps into the four copepod production tanks, thereby feeding the copepods.

Nutrient supply (6):
Two pumps are installed, one to each bioreactor, to supply the bioreactors
with nutrients. The
nutrient is

prepared in sterile blue cap bottles and has no contact with the users,
preventing contamination. The nutrient recipe used is B1 media, solution A (inorganic nutrients)
mixed with solution C (vitamins) (P. J Hansen 1989). Th
e B1 media can be supplied form the
pumps with 0.1


1.5 l/hr.


Light

(
7
)
:

Optimal growth can be achieved using continuous light at an intensity of up to 10,000
lux when using continuous, strong light within the Photosynthetic Active Radiation range.
LC
D

light
s

are installed that
can be dimmed

to three different intensities. The advantage with LCD light
is that they

do not heat the water, and are cost effective.
T
he t
wo

algae bioreactors can be

individually
regulated.




IMPAQ Indoor RAS manual

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7


Experimen
s, results and recommen
dations

Here I need input from Minh and Claire’s work!

Light conditions for culture

Aims:
Adjusting the light during the time to prevent inhibition (at low concentration) and light
limitation (at high concentration)



Understanding the absorption and scattering



By the algae cells


-
R.baltica

will be cultured in batch in 5
-
10 L glass bottles to reach the highest densities
(≈ 2
-
3 x 10
6

cells/mL)


-
Make a dilution series of the algal culture


-
Measure the light in the
cultures with difference densities: 3x10
6
, 2.5x10
6
, 2x10
6
, 1.5 x10
6
,
1 x10
6
, 0.5x10
6

cells/mL


-
Corellation between the light absroption and the density of the algae (in the photobioreactor
the density of algae can reach much higher than
3x10
6
cells/mL in f
act we aim at 10
7

cells/ml)



By the glass material of the
cylinders



Calculation to model the light condition in the photobioreactor
(Søren LN)

Principle for culture conditions CO
2

(pH)

Aims:
Adjusting pH by the addition of CO
2

(sufficiently)

Start the photobioreactor with the 10 L algae cultures at the
mid
-
exponential growth phase.



At start:

-

the initial concentration of algae in the photobioreactor is app. 200 000 cells/mL.

-

Without CO
2

supply

-

Record the density and pH of cultures



Follow the
growth of algae until they reach the stable phase



Start supplying CO
2

to adjust the pH

-

1 cylinder at pH 8.0

-

1 cylinder at pH 7.5

-

Record density

IMPAQ Indoor RAS manual

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8


0
0.1
0.2
0.3
0.4
0.5
0
5000
10000
15000
20000
25000
30000
35000
Specifc growth rate ug C L
-
1

R. salina
[Cell ml
-
1
]

Berggreen et al. (1988)

Measurements on algae: When we have the system up running and steady
-
state production is
obtained




Concentration

of algae (Coulter coulter)



Size of algae (Coulter counter)



Biochemical content of algae



Carbon content (CNA analyzer)



Nitrogen content (CNA analyzer)



Fatty acids (GC/MS)



Amino acids (HPLC)



Chlorophyll a content (Spectrophometry)

Algae feeding system

Berggr
e
en et al (1988) has shown that it is important to ensure feeding above 20,000 cell ml
-
1

when
the ambition is to obtain maximum specific growth and egg production tae of
A. tonsa
.








To
ensure the feeding criteria described by Berggreen et al. (1988), a series of experiments
estimate
d

the specific l
oss of algae from the tanks over

time
.

The variabl
es are c
oursed by
algae
sedimentation
and water

exchange in the production tank
. The tanks were induced with a known volume of
Rhodomonas
salina

to the copepod

culture tanks, following concentrations above feed limitation.


Batch feeding
:
Initial high
concentrations of algae were

monitored
over at least 24h.
24h

is the
normal practice when feeding copepods in most reported cultures. There were
NO

copepods in the
tanks, so the only variable were in
-

out
flow velocity into the tank. Not surprising did we l
oss less
Figure
4

Graph modified from Berggr
e
en et al. (1988). Specific growth rate as a
function of cell ml
-
1 of
R. baltica
.

IMPAQ Indoor RAS manual

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9


0
5
10
15
20
25
30
35
40
45
0
5
10
15
20
25
30
Chlorophyll a µg L
-
1

Time (h)

Batch feeding

0L hr-1
10L hr-1
40L hr-1
100L hr-1
300L hr-1
0
5
10
15
20
25
30
35
40
0
5
10
15
20
Chlorophyll a µg L
-
1

Time (h)

Continues feeding

40L hr-1 inflow +
0,8L hr-1 pump
10L hr-1 inflow +
0,4L hr-1 pump
algae at the static situation with no
inflow of water, this is thereby the
isolated effect of sedimentation of
algae we obtain. And when water
velocity of the inflow were increased
the loss of algae from the tank
increased. Thereby we from this fi
gure
can estimate effects of sedimentation

(1% h
-
1
)
, loss effects to the tank
outflow and the combined effect
of
these.
The
conclusions

are

that batch
feeding

is a good strategy whit no
water exchange, but with water
exchange it is recommended either to
increase the intensity of batches over a
day, or apply continues feeding.


Continuous feeding
:
An experimnet
was setup with
same experimental
procedure as with batch feeding in
regards algae initial concentrations,
time and
measurement methods
.
In

regard of flow only 0, 10 and 40L h
-
1

were used since with higher flows the
loss rate of algae are to high,
especially if grazing is applied to the
equation. A exponetial model was
fitted to the batch feeding result for
the three choosen flows and the
equ
ation were used to calculated the
compensatory amount of algae that
has to be added into the tank. An
initial dose

of algae
were added,
similar to the the one used in the
batch experiment
,

thereby obtaining

feed in excess for copepods.
Thereafter algae are

continuesly
supplied into the tank with a
Figure
5
.

R
esult
s

from the experiment with measured
values of chl a the loss to sedimentati
on and outflow of
water from t
he tank, at different tank outflow water
velocities
.

Figure
6

Continues feeding. A batch of algae spikes the
tank to initial high concentrations; hereafter
concentration levels are kept by continues inflow of
algae from a peristaltic pump.

IMPAQ Indoor RAS manual

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10


peristaltic pump set to a
flowrate co
m
pensating for sedimentation and outflow of algae from the
tank, at the different flow rates
.

The results showed that at the chosen flow rates it is indeed possible to compen
sate for the loss of
algae due to sedimentation and outflow of algae from the tank. This results in keeping a feed level
for copepods in excess.
Conclusions

are that with simple calculation and flow control
,

algae can be
supplied as food in excess for cop
epods.

Acartia tonsa

has a specific ingestion rate of maximum 1.
3

d
-
1

at the optimal growth rate 0.44 d
-
1
.

Thereby
the feed requirement is

a total of ~1000 µg C L
-
1
.
The Chl a: Carbon ration for
R. salina
is
around 30. Thereby 34ug Chl a equals the dietary need for
1
copepods d
-
1
.





































Equation
1

Equation 1 is used to calculate the daily need of feed for the copepod culture. When
the total daily
chl a ingestion is known then it can be compensated by adjusting the inflow of feed from the
feeding pump.

Combined with the different compensation rates shown on figure
6
then
the ideal
copepod feeding scheme can be setup.


IMPAQ Indoor RAS manual

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11


Copepod rearing

1.

Aim of Co
pepod rearing facilities

The objective is to combine RAS from known aquaculture systems and apply it to copepod cultures. And to
optimise the system design to a high biomass production system that can provide an economical competi
tive
live feed system.
The main aims of the study are as following:



Obtain high copepod stocking densities



Obtain high egg productions



Apply labour efficient culture management



Develop and modify culture system


Approach of the project

A

novel Recirculated Aquaculture System (RAS) has been developed and modified to counter the challenges
when cultivating calanoid copepods intensively. The calanoid copepod
Acartia tonsa

is the specie used, since
it is an excellent and well documented model

organism. Earlier study with
Acartia tonsa

has shown that their
eggs can be stored for prolonged periods, and from the eggs, nauplii can be hatched and used for live feed to
marine fish larvae. This provides a two way system. Either the technology from th
is project can be directly
transferred to the marine fish farms (De
-
central), or a central egg production facility can supply eggs for live
feed to the marine fish farm (Central). So the farmer has the option to choose between central versus
decentralised
production of copepods eggs, adding flexibility to the system. RAS equipped with sensors and
controlled by Programmable Logic Control (PLC) is used since it is a labour efficient system. Different
challenged in the project will be solved with everything f
rom fully controlled laboratory setups to big scale
experiments in the total RAS.

The goal is to effectively have high intensive copepod cultures, still with a high egg production
yield, as cost effective as possible.



IMPAQ Indoor RAS manual

Page
12


Fact box
-

RAS equipment list
:

Multi Function Bag Filters
(
5µm
, enviro EBF009
-
7
)

Pump sump with 2 x IWAKI MX250CVSE pumps.

1 x
AQUAFLOTOR

type AQ 300, Protein skimmer.

1 x
Water Biofilter 4 chamber version ( RK
BioElements, area pr.

m
3

= 750m
2
, weight mass = 0,93
g/cm
3
).


1 x WUVX 40


UV
(WATER Aps 40W)
.

4 x 320L HDPE black Copepod tanks (
ɸ

= 760mm) with
an intake flow meter regulated from 10 to 300 L h
-
1
.

4 x OxyGuard 4
20 Dissolved Oxygen probe.

1 x OxyGuard PH MANTA (0


14⁰ ⤮

1⁸ 佸O䝵慲a⁳慬楮楴i⁰牯b攮

Copepod culture design

Conceptu
al
designs of the intensive copepod system custom build

for the IMPAQ project.


Figure
7

conceptual drawing of the Recirculated Aquaculture System

(RAS)

installed
at
RUC
-
ENSPAC

for use in
the IMPAQ project.

Description
of RAS

In the Recirculated Aquaculture System at
RUC t
he water is pumped around in the
system in two loops. One loop is a cleaning
loop w
h
ere the water is purified in different
steps.

First the intake water to the system in
filtered through a 5
µ
m bag filte
r, this water
enters into the 2m
3

pump sump. From here
the water is pumped in a flow that can be
regulated from 300L h
-
1

to 3000 L h
-
1

depend
ing

on how dirty the water is, into
the protein skimmer were proteins are
removed from the water by injecting micro
bubbles of air into the protein skimmer.
Automatic cleaning of the protein skimmer
by freshwater are installed to

flush out the
IMPAQ Indoor RAS manual

Page
13


0
500
1000
1500
2000
07/Jan
11/Jan
15/Jan
Eggs tank
-
1

Acartia tonsa eggs

collected prote
ins. After the protein skimmer the water is by gravity running into the 1m
3

four
-
chamber biofilter. The biofilter is filled with
bio media
. There is a down flow in the first biofilter
chamber, up flow in the second, down flow in the third and an up flow in

the last chamber before
the water returns to the 2m
3

pump
-
sump. Inside the pump sump both heating and chilling are
installed to regulate the temperature of the water.

The other flow loop in the system is pumping the water through another 5
µ
m bag filter
,
a UV and
then into the copepod tanks. From the copepod tanks the dirty water flows back into the pump sump
were it re
-
enters the cleaning loop.



The design of a copepod tank has to facilitate easy access

for culture management. Tanks
hydrodynamics
has

to be optimized
to keep animals and eggs inside tanks, not disturbing grazing
,
facilitate high
copepod
densities

etc.

Copepod densities

A number of experimental trials have been performed and are in progress:
Daily measures of the
population were monitored
to find the culture density
. A
n

insert in the tank was installed with a
false bottom consisting of a 200 µm screen. The idea with a screen was to separate
the target
species, the calanoid copepod
adult
Acartia tonsa

from eggs and nauplii, preventing cannibalism
and easing egg cleaning

(see figure
8
)
.


Figure
9
.

P
opulation development of
Acartia tonsa

inside the
200µm mesh
insert of the copepod tank.



0
20
40
60
80
100
120
140
160
180
200
07/Jan
12/Jan
17/Jan
22/Jan
Individuals (L
-
1
)

Acartia tonsa


Nauplii(L-1):
Copepods(L-1):
Figure
8
.

The
design of a
copepod tank with a 200µm
mesh screen inserted.







Inflow

Outflow

Figure
10
.

Acartia tonsa eggs
harvested from the production
tanks.

IMPAQ Indoor RAS manual

Page
14


After 3

weeks the population in the
insert

declined to a minimum level and the experiment stopped
.

The conclusion is that a
n

insert prevent recruitment for the adult population and will function
well

in a batch culture system
,

but for a
continuous

RAS it
appears

not
to be
the optimal

solution
.
Further the screen kept clocking with air bubbles and algae, and daily caretaking was necessary,
which is not practical in a low labour automatic copepod system.

Also

determining
the copepod
population above and below the screen is impractical since two di
fferent not comparable methods
has to be used.

For tank cleaning the screen has to be removed which is impractical and will
potentially increase mortality for the copepods since handling of copepods can enhance mortality
(Jepsen et al. 2007).

The total ha
rvest of eggs from
A. tonsa

did

n
o
t meet the expectation according to the number of
adult
s

present in the production tank. Only 20% of expected eggs were harvested from the system.
Eggs were lost in the system and further experiment
s

will optimise egg harv
est from tanks.

To investigating the harvest of
A. tonsa

eggs from the bottom drain, we first monitored different out
flow´s from the bottom drain. It was quickly obvious that maximum flow was required, which we
also expected (data not shown). Therefore a
nother experiment investigated the amount of water
harvested at maximum flow to yield most possible eggs from the bottom drain (see figure xx).




Figure
11

Acartia tonsa

eggs

harvested as a function of
litres

of
water flushed

out of the tank

The bottom drain samples were collected in successive flush
es

but the first sample had a higher
quantity of eggs. From figure
11

can be seen that flushing more than a quick first flush do not
harvest a lot of extra eggs. We harvest the eggs near the bottom drain and draining a lot more water
from the tank will not increase our egg harvest from the tanks. Therefore we must apply oth
er
methods to harvest eggs stocked other places in the tanks. Further experiment will investigate a
n

effective harvest system

like e.g. applying a brush
, mechanical filter etc.


0
50
100
150
200
250
300
350
400
0
1
2
3
4
5
6
7
8
Eggs (ind L
-
1)

Volume tapped from the tank (L)

Eggs per Liter at different outflow volumes

IMPAQ Indoor RAS manual

Page
15


Egg harvest and egg outflow experiment.

To quatify the egg harvest during a da
y a copepod production tank were stocked with 300,000 eggs.
The water in
-

and out
flow
was
10 L/h
, and the eggs lost to the outflow, and hatched inside the tank
together with harvested 24h after stocking with eggs were estimated.

The fate of the eggs
with

a in
-

and out
flow of 10L h
-
1, and an initial stocking of 300,000 eggs is
presented in figure
12
.















The results from the experiment
with a
n in
-

and outflow of 10L h
-
1 showed that, o
ptimized harvest
method

has to be
applied
. We experience that 122,000 eggs
was
unaccounted for, and these should
potentially still be
available for harvest.

Further we had a

daily loss of
18,000 eggs and 44,
000
nauplii from the top outflow, therefore
pre
-
screening of the outflow has to be installed
, in future
setups
.

Copepod culture management

Critical elements


T
able

1:
H
ighlight
s of

biological features of

Acartia tonsa

there

usage

for aquaculture
, and
recommendations for intensive cultures of

A. tonsa
.

Biological features of

Usage

for aquaculture

Recom
mendations for
Acartia tonsa

cultures

Wide temperature range from
-
1 to 32°C
(Paffenhofer and
Stearns 1988; Chinnery and


Cultures can be adapted to lo
cal
temperature
conditions



Eggs can be cold stored



17 to 25°C, for
optimal egg
production and
development time

Inflow 10L h
-
1

Lost from Top outflow d
-
1

18,000 eggs d
-
1

44,000 nauplii d
-
1

Harvested from

Bottom

outflow d
-
1

30,000 eggs d
-
1

1,000 nauplii d
-
1

Initiated

with 300,000 eggs

Hatching success
24h
: 43.4 ± 6.4%

Internal los
t
:

122,000 eggs

Figure
12

shows fate of addition of 300000 eggs with an in
-

and outflow of 10L h
-
1
.

IMPAQ Indoor RAS manual

Page
16


Williams 2004
; Hansen et al.
2010).


Wide salinity range 5 to 36 ‰,
tolerate rapid salinity change
(Cervetto, Gaudy et al. 1999;
Chinnery and Williams 2004;
Hojgaard, Jepsen et al. 2008;
Ohs, Rhyne et al. 2009)



Cultures can be adapted to local
conditions



Salinity change can be used to
suppress invasive pathogenic and
other nuisance organisms



Abrupt salinity change can be
used to store eggs




From 30 to 36‰ for
cultures, less energy
used for
osmoregulation.
(Lance, J., 1965
).



For eggs storage
transfer from ambient
culture salinity to
milliQ water
(H
ø
jgaard, Jepsen et
al. 2008)


Egg production
0.4

to
55

eggs
female
-
1

day
-
1
(Stottrup,
Richardson et al. 1986
;
Jepsen,
Andersen et al. 2007
;
Medina
and Barata 2004
;
Peck and
Holste 2006
;
Drillet, Jepsen et
al. 2008)



High number of nauplii day
-
1
from batch or continues cultures



Eggs can be harvested and cold
stored and used as
back up of live
nauplii production



20 egg female
-
1

day
-
1

should be minimum
production
expectations

Light regimes for
nauplii

to
adult from 0L:24D to 12L:12D
(Peck and Holste 2006)

Light
regimes for eggs 12L:12D
(Peck and Holste 2006)



Cost for artificial light above
cultures can be saved



Darkness can be use
d to suppress
invasive organism



Some reports about light
influence hatching success
therefore recommended regime
for eggs



UV
-
radiation can be used to
enhance copepod pigmentation,
and thereby visibility for
predator.



Optimal feed uptake
and thereby egg
p
roduction in
darkness
(Stearns,
Tester et al. 1989)




UV
-
radiation
as
pigment manipulator
(Hansson 2000)



Body size and somatic growth
can be regulated by
temperature
(Ambler 1985;
Chinnery and Williams 2004;
Hansen, Drillet et al. 2010)




Suitable size ranges can be
“constructed” for different types
of marine fish larvae




Smaller
cephalothorax size
with higher
temperature
(Hansen,
Drillet et al. 2010)


NH
4
/NH
3
levels from 0,
03

to
0,4
7

mg L
-
1
, with no observed
effect on cultures
(Sulli
van and
Ritacco 1985
;

Buttino 1994
;
Jepsen et al. 2013
)




Maximum stocking density for
batch cultures can be calculated



Cultures can be maintained with
biofilter
technology



Keep concent
rations
below 0
.
03 mg NH
3

L
-
1

for nauplii and
below 0.
4 mg NH
3

L
-
1
for Adult
A. tonsa
.

IMPAQ Indoor RAS manual

Page
1
7


Fact box
-

Acartia tonsa

eggs definitions:

Subitaneous

e
ggs

= eggs that hatch within 72 hours from produced, at 17°C.

Quiescent eggs

=
subitaneous

eggs that with a change of the physical
condition can be provoke into arrested devel
opment, and be awakened again
when conditions is returned to normal.

Diapause
e
ggs
= Eggs that has to go through a refractory phase before they
can hatch.

Delayed Hatching eggs

= Eggs that are maternally determined to hatch at a
predetermined time poi
nt


Oxygen levels

above
2
.
0mg

O
2

L
-
1

(Marcus, Richmond et al.
2004
;
Sullivan & Ritacco,
1985)




Ca
n tolerate low levels of oxygen




Pressurised

O
2

is not necessary
for maintaining cultures



Eggs are not negatively affected
by anoxia



Temperature difference and
oxygen is not a problem



Keep oxygen levels
above 2 mg O
2

L
-
1
.

pH level from 7.
7 to
9.5

(Sullivan and Ritacco 1985
;
Hansen et al. in prep.
)



Can survive a wide range of pH
without any effect



pH can be regulated to maintain
other abiotic factors steady in
cultures



Eggs are not affected by high pH



Keep pH below 9.0
to
ensure no effect on
egg production
, egg
hatching,

and
copepod (especially
nauplii)

mortality


Fast generation time from 14 to
19 days
(Chinnery and
Williams 2004; Drillet, Jepsen
et al. 2008)




Selection



Restocking of cultures not critical



Fast adaption to environmental
factors



Physiological plasticity



Selection of large
males will optimize
the female’s egg
production
(Ceballos
and Ki
ø
rboe 2010)

Egg storage for up till 1 year

(Drillet, Iversen et al. 2006
;
Stottrup, Bell et al. 1999)



Valuable tool for storage and
supply of nauplii to fish larvae



M
aximum storage
time at 5
°
C and
anoxia is 1 year


Feed

(Berggreen, Hansen et al.
1988)



Can enhance egg production



Can be used to biochemically
enrich copepods.




Rhodomonas salina

in
excess
10
00 µg C L
-
1

Correct size
range, go
o
d
biochemical
profile



Density
depend egg production

A key feature to
optimise the production
in the RAS is to
maximi
z
e eggs
produced per individual.
Earlier studies have
shown that eggs of
A.
tonsa

can be stored for
up till one year, still
keeping a valuable
biochemic
al profile
(Drillet et al. 2006
).
IMPAQ Indoor RAS manual

Page
18


Quiescent

A. tonsa

eggs provide a product that can provide the aquaculture industry with a live feed
product, similar to
Artemia

(brine shrimp)
cysts. Since eggs easily can be transported around the
globe and hatched and feed out to marine fish larvae.
One solution to
optimize
mass production of
A.
tonsa

eggs is by increasing individual stocking density L
-
1
. In the literature it seems that no one has
stocked calanoid copepods above 2000 ind.L
-
1

in their experimental facilities, therefore this upper
limit
ation was worthwhile to challenge if live feed production based on copepods shall be
commercially interesting. In the IMPAQ project we tested in a small scale laboratory set
-
up the egg
production of
A. tonsa

at densities ranging from 10 to >5000 ind. L
-
1

a
nd followed the hatching rate
of the produced eggs. With the ultimate ambition to maximize the calanoid copepod densities in
intensive mass cultures for live feed, where the egg harvest of subitaneous eggs is optimized
without stimulating an eventual
delay
ed hatching
egg production
.


Figure
13
.

Acartia tonsa

total egg production harvested per litre of culture per day

(Drillet et
al
.

in prep.).

In figure 12 a yield of 10000 eggs were
achieved with densities
around

~1000 individuals L
-
1
.
In
this experiment there was not applied any water exchange and thereby the result is the combined
effects of chemical and physical density upon egg production. This resulted in an accumulation of
inorganic nutrients excreted from t
he copepods as a function of time as shown in figure
13
.
The
conclusion is

that with batch cultures with combined effects of density and inorganic nutrients the
maximum stocking density is ~1000 Ind. L
-
1
.



0
2000
4000
6000
8000
10000
12000
14000
0
1000
2000
3000
4000
5000
6000
Total egg production [eggs L
-
1

d
-
1
]

Densities [Ind. L
-
1
]

IMPAQ Indoor RAS manual

Page
19



Figure
14

shows
accumulation of TAN, NO3 and NO2 over time at different copepod stocking
densities (D
rillet et al in prep.
).

To investigate the effect of inorganic nutrients another study was setup (see table
2
).

Table
2

from Jepsen et al
(
2013).

pH

NOEC adult
[µgNH3 L
-
1
]

LOEC adult
[µgNH
3

L
-
1
]

Adult density
[Ind. L
-
1
]

NOEC nauplii
[µgNH3 L
-
1
]

LOEC
nauplii
[µgNH
3

L
-
1
]

Nauplii density

[Ind. L
-
1
]

7.5

477

1,789

170* 10
6

30

81

10.8*10
6

8.0

477

1,789

55*10
6

30

81

3.5*10
6

8.5

477

1,789

18*10
6

30

81

1.2*10
6

9.0

477

1,789

7*10
6

30

81

444,223

9.5

477

1,789

3.4*10
6

30

81

213,262


The water quality study showed that the earlier observed maximum yield of eggs were only an
effect of density and not of inorganic nutrients. Although it verified that it is important to keep track
IMPAQ Indoor RAS manual

Page
20


of inorganic nutrients over time and with its pH dependent

equilibrium, increased pH will result in
more toxic environment for the copepods.
It is recommended that pH and inorganic nutrients
are monitored weekly, especially for batch cultures of copepods. For high intensive culture
this is a daily task for the fa
rm manager, as what is normal practices in aquaculture facilities.


Economic evaluation

In this research, we are going to explore the economic feasibility of intensive copepod production
for commercial scale. Data from Roskilde University lab experiment

are used. Cost benefit analysis
is employed (without incorporating the monetized values of benefits of copepod on the entire food
chain). We assumed that the benefits of the copepods on the entire food chain are reflected on their
price. The result reveal
s that intensive copepod production for commercial scale can actually
payoff.


Results

Input from Tenaw

Conclusions and future studies

From these preliminary studies we can conclude that algae production
… input MINH/Claire


When feeding algae to copepods

it is important to apply continues feeding instead of batch feeding.
It is indeed possible to counteract both algae sedimentation and water exchange from copepod
rearing tanks with simple calculations. The foundation for these calculations has been invest
igated
and ready to apply for intensive copepod cultures. In regard of densities a preliminary limit were
found of ~1000 ind. L
-
1
.
This will be further investigated in experiments removing effects of
inorganic nutrients together with volume effects.

Litera
ture study together with experiments found
the basis for cultures of the copepod
Acartia tonsa

and will be utilized for general recommendations
of the caretaking of this animal.

I
nitial experiments in the RAS showed a loss of eggs, nauplii and
copepods fro
m the production tanks. Therefore the RAS are modified with new design of outflow
together with optimised harvest methods, and future results will show the effort of these studies.


Economics… Tenaw.