Aquaculture Research, 2012, 43, 91–98
doi:10.1111/j.1365-2109.2011.02807.x
Semi-continuous mass culture of rotifers (Brachionus
plicatilis) using an automatic feeder
Venetia Kostopoulou1,2, Manolis Vasilakis1 & Pascal Divanach1
1
Hellenic Centre for Marine Research, Institute of Aquaculture, Heraklion, Crete, Greece
Department of Veterinary and Fisheries, Prefecture of Heraklion, Heraklion, Crete, Greece
2
Correspondence: V Kostopoulou, Hellenic Centre for Marine Research, Institute of Aquaculture, PO Box 2214, Heraklion, Crete 71003,
Greece. E-mail: vkostop@biol.uoa.gr
Abstract
This study describes the rotifer mass culture protocol
developed at the Institute of Aquaculture (HCMR) in
Crete. Rotifers were cultured semi-continuously at a
density of 257.6 2.6 ind. mL 1, with a daily dilution rate of 25%. An automatic feeder was used to ads
minister dry feed (Selco Sparkle ) every 10 min. A
number of factors were investigated so as to provide
an in-depth analysis of the advantages and disadvantages of the two systems, especially when compared
with traditional protocols such as batch culture. The
use of these two systems resulted in a culture of considerable duration (41 days) and daily yield
(0.21 0.01ind.106 day 1 L 1), using exclusively
dry feed. Both systems can be easily applied in hatcheries, requiring few modi¢cations on existing protocols and contribute to a stable and predictable
production. Further improvements include manipulation of the dilution rate, food ration, culture volume
and duration, as well as choice of a phytoplanktonbased diet.
Keywords: aquaculture, hatcheries, live feed,
feeding
Introduction
The rotifer Brachionus plicatilis (Mˇller 1786) is used
as ¢rst feed of ¢sh larvae in aquaculture farms. It is
considered to be an indispensable element of mass
production, due to its size, ease of culture, high
growth rate, slow mobility and easily adjusted nutritional pro¢le. On the other hand, rotifer mass production is also regarded as one serious source of troubles
© 2011 Blackwell Publishing Ltd
in intensive rearing of ¢sh larvae, mainly due to
discontinuities in its culture (crashes), variable nutritional pro¢le and overall condition.
Control over rotifer mass culture has been
attempted using di¡erent approaches. From a biological standpoint, a number of indicators (egg ratio,
swimming speed) provide information on the status
of rotifer culture. On a technological basis, production techniques have improved by increasing the sophistication of culture systems. The simplest system
is batch culture. In this system, rotifers are allowed
to grow with addition of food for a speci¢ed period of
time, after which they are harvested, in order to be
o¡ered as feed. The highly variable conditions that
prevail during batch culture create an unstable
environment for rotifers. In the semi-continuous system, rotifer density remains constant through periodic, partial harvesting of the culture. In this way,
the culture medium is replenished, and any metabolic products are diluted. This type of culture can
be maintained for longer periods of time, because it
creates a more stable environment for the cultured
organism. Other systems of increasing complexity
are the continuous culture and high-density system
(Dhert, Rombaut, Suantika & Sorgeloos 2001;
Lubzens, Zmora & Barr 2001; Conceicao, Yu¤fera,
Makridis, Morais & Dinis 2010).
The administration of food is another basic parameter a¡ecting rotifer mass culture stability. According to Olsen (2004), a common problem in rotifer
mass culture is overfeeding, which may, in turn, result in bad water quality and invasion by competing
microzooplankton (e.g. ciliates). Therefore, control
over the administration of the daily food ration is expected to have a positive e¡ect on culture stability.
91
Semi-continuous rotifer mass culture V Kostopoulou et al.
Aquaculture Research, 2012, 43, 91–98
Despite the major advancements in know how,
most hatcheries still use simple, but ine⁄cient rotifer
mass culture protocols, such as batch culture (Dhert
et al. 2001). Sophisticated systems have been proposed (e.g.Yoshimura, Usuki,Yoshimatsu, Kitajima &
Hagiwara 1997) but not widely applied in production
up to now. It seems that hatcheries call for simple
modi¢cations in existing mass culture protocols that
can be easily applied based on present knowledge. In
addition, food is usually hand administered in predetermined doses, several times per day but restricted
by night.
This paper presents two simple systems ^ semi-continuous culture combined with automatic feeding ^
that could lead to an improvement of rotifer culture
stability and workload, requiring few modi¢cations
on traditional protocols. It provides an in-depth
analysis during real-time rotifer mass production
followed at the Institute of Aquaculture (HCMR)
in Crete, Greece. This paper is an e¡ort to
make automation accessible and easily applicable to
any hatchery.
S/Mill, ATAGO CO LTD,Tokyo, Japan). pH and oxygen
were monitored daily (HQ40d, Hach, Dusseldorf,
Germany). Total ammonia was measured at regular
intervals using a test kit (Tetra, Melle, Germany). Unionized ammonia was calculated according to the
corresponding values of pH, salinity, temperature
and total ammonia (Bower & Bidwell 1978). No light
was added and all cultures were subjected to natural
sunlight (0^200 lx).
Rotifer samples (200 mL) were collected daily
(09:00 hours) and two 1mL sub-samples were
counted under the stereoscope (Olympus SZ40,
Olympus, Hamburg, Germany), in order to obtain rotifer density. The growth rate (r, ind. day 1) was estimated separately for the two phases of production:
Materials and methods
Rotifers (B. plicatilis s.l.) were mass cultured in cylindro-conical tanks (1.6 m3 capacity; n 5 84) for 14
months, from October 2008 until December 2009.
They were introduced in the tanks at a starting volume of 1000 L. Every day, the volume of the rotifer
mass culture was increased, so as to reach 1600 L
after, on average, 4 days (Table 1). This preliminary
phase aimed at increasing rotifer numbers through
successive culture medium addition. It corresponds
to a batch-type culture system, and will be referred
as such from here on. Once the mass culture tanks
reached maximum capacity (1600 L), the culture
protocol changed from batch to semi-continuous.
The latter represents the main culture phase, during
which part of the culture (400 L) was replaced daily,
resulting in a constant ¢nal volume (1600 L). The
rotifers harvested during the daily removal of part of
the culture were used as feed for ¢sh larvae. The two
phases ^ preliminary and main ^ represent di¡erent
culture systems ^ batch and semi-continuous,
respectively, hence they will also be studied separately and compared. Care was taken to keep the
main physicochemical parameters constant, whenever possible. Temperature was controlled continuously by means of sensors placed inside the tanks.
Salinity was checked every day (refractometer Atago,
92
rbatch ¼ ðlnQn lnQn1 Þ=t
where lnQ is the natural logarithm of rotifer total
quantity, n corresponds to the day n and t, to the
duration of culture (days).
rsemi-continuous ¼ ln ð1 DV=VÞ
where DV represents the fraction of the tank volume
(L) replaced at each dilution and V the total culture
volume (Navarro & Yu¤fera 1998a). The daily yield (Y,
ind.106 day 1 L 1) was calculated as follows:
X
t DV batch
Y batch ¼ ðQf Qi Þ=
where Q corresponds to the total quantity of rotifers,
f represents the ¢nal day of culture, i the initial
day of culture, t, total culture duration (days) and
DVbatch represents the di¡erence in volume between
initial (i) and ¢nal (f) days of culture (L). In the
case of the semi-continuous culture, the daily
yield corresponds to the quantity of rotifers removed
from each tank daily divided by DVsc, which
is the fraction of the tank volume (L) replaced at each
dilution. The e⁄ciency (E, %) was estimated by adjusting the formula reported by Navarro & Yu¤fera
(1998a, b):
E¼
rotifers produced daily ð106 day1 Þ rotifer dry weight ð106 gÞ
daily amount of feed in dry weight ðg day1 Þ
100
The dry weight of rotifers and their feed was
determined by drying samples (n 5 3) at 70 1C to
constant weight.
The rotifer populations were also monitored in
terms of residence time in the mass culture tanks
during the main production phase. It is common
practice in hatcheries to use part of the harvested rotifers as inoculum for the following mass culture. In
such a case, successive mass cultures are conducted
with the same rotifer population. In this study, each
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 91–98
Semi-continuous rotifer mass culture V Kostopoulou et al.
Aquaculture Research, 2012, 43, 91–98
Table 1 Management of rotifer mass cultures
Main phase
(semicontinuous)
Preliminary
phase (batch)
Volume (L)
Initial
Final
Fraction replaced daily
Initial population (ind. 106)
Culture duration (days)
Mean SE
Minimum
Maximum
Total
Temperature ( 1C)
Salinity (g L 1)
PH (mean SE)
Oxygen (mg L 1)
Total ammonia (mg L 1)
Mean SE
Minimum
Maximum
Unionized ammonia (mg L 1)
Mean SE
Minimum
Maximum
1000
1600
P
1600
400
158.09 7.87
4.23 0.17
2
8
12.00 0.80
4
38
41
25
25
7.35 0.003
45
25
25
7.58 0.013
45
1.67 0.36
0.50
3.00
45.00
5.00
45.00
0.024 0.004
0.01
0.04
40.05
0.04
40.05
The test kit does not detect levels above 5 mg L 1 with accuracy (n 5 84).
Po0.001.
SE, standard error.
rotifer population that was introduced in the mass
culture tanks from stock cultures was assigned a
code (lot number) and was followed throughout its
mass culture (multiple culture tanks) until a new inoculum was introduced.
s
Rotifers were fed Selco Sparkle (INVE S.A., INVE,
Ghent, Belgium). The quantity of food added in the
tanks was not constant, but was based on the daily
measurement of rotifer density (Table 2). The addition of the feed was accomplished using an automatic
feeder, which allows the addition of food at regular
intervals without human interference. The automatic feeder is composed of a dispenser (3-L capacity), which is placed on a rotating axis, above the
centre of each tank. The daily total quantity of food
is introduced into the feeder and the frequency of administration is set by the user. In this case, food addition in the tanks was programmed every 10 min by
elemental doses (which can be calculated if the daily
total feed quantity is divided by144), allowing emptying of the reservoir twice a day.
The data were subjected to statistical analysis. Culture duration, pH, daily yield and e⁄ciency were compared between phases with the non-parametric test
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 91–98
s
Table 2 Calculation of feed quantity (Selco Sparkle , g 106
rotifer 1) added daily in mass culture tanks based on rotifer
density (ind. mL 1)
Rotifer density (ind. mL 1)
Daily feeding rate (g Selco
s
Sparkle 106 rotifer 1)
0–90
100
150
175–200
250
300
350
400
450
500
1
0.65
0.60
0.55
0.44
0.39
0.34
0.33
0.31
0.30
Mann^Whitney. One-way analysis of variance (ANOVA)
was used for the comparison of lot number with a series of parameters. One-way ANOVA was also performed
on square-root transformed rotifer density data. Comparison of means was conducted with the Tukey HSD
Statgraphics Plus 5.0, Statistical Graphics Corporation, Warrenton, Virginia, USA. Di¡erences were considered to be signi¢cant at Po0.05.
93
Semi-continuous rotifer mass culture V Kostopoulou et al.
Aquaculture Research, 2012, 43, 91–98
Table 3 Mean standard error of main parameters calculated for the two phases of rotifer production
Growth rate (ind. day 1)
Daily yield (ind. 106 day 1 L 1)
Efficiency (%)
Preliminary phase (batch)
Main phase (semi-continuous)
P
0.30 0.01
0.11 0.01
9.41 0.41
0.29
0.21 0.01
9.01 0.41
–
NS
Po0.001.
NS, non-signi¢cant.
Figure 1 Mean 95% Tukey HSD intervals of rotifer density (ind. mL 1) with time (days). Means with di¡erent letters
are statistically di¡erent (Po0.001).
Table 4 Characteristics of residence time of rotifer populations in mass culture tanks (for explanation, see ‘Materials and
methods’)
Lot number
Total number of mass culture tanks
1
2
3
4
5
6
7
37
53
6
14
3
2
2
Results
The two production phases showed similarities and
di¡erences. The growth rate (r) and the e⁄ciency (E)
did not di¡er between phases, whereas the daily yield
(Y) was statistically higher (Po0.001) during the semicontinuous phase, compared with the batch (Table 3).
Rotifer density showed temporal variation (Fig. 1).
There was a gradual increase during the ¢rst days of
culture (mean standard error): 168.05 6.63 ind. mL 1 on day 0, 215.42 8.12 ind. mL 1 on day 1
94
Start date^end date (day/month/year)
12/09/2008–28/04/2009
27/03/2009–05/10/2009
19/09/2009–23/10/2009
18/10/2009–07/12/2009
26/11/2009–16/12/2009
03/12/2009–19/12/2009
04/12/2009–12/01/2010
Total duration (days)
229
193
35
51
21
17
40
and 259.96 9.10 ind. mL 1 on day 2. Rotifer density
remained stable thereafter (265.50 2.52 ind. mL 1)
until the 25th day, after which a decreasing trend
was evident (220.24 5.61ind. mL 1). Therefore, an
increase in volume during batch culture corresponded
to a concomitant increase in rotifer numbers, followed
by daily dilution during the semi-continuous culture,
which had a stabilizing e¡ect on rotifer density.
Parallel growth of ciliates was also monitored.
A range of 0^2670 ind. mL 1 was recorded during
production.
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 91–98
Aquaculture Research, 2012, 43, 91–98
Semi-continuous rotifer mass culture V Kostopoulou et al.
Figure 2 Mean standard error of daily yield (Y, ind.106 day 1 L 1) of semi-continuous culture with lot number (for
explanation, see ‘Materials and methods’). Means with di¡erent letters are statistically di¡erent (Po0.001).
The rotifer population of the mass culture tanks
was renewed seven times from stock cultures during
the period of 14 months of mass production (Table 4).
The residence time of rotifers in the mass culture
tanks was not of equal duration, but showed a tenfold
variance (from 17 to 229 days). When the lot number
was compared with a series of parameters, it showed
signi¢cant di¡erences with the daily yield during the
semi-continuous phase of production (Fig. 2). Namely,
the daily yield was signi¢cantly lower (Po0.001)
in the lot numbers of longer duration (1 and
2:0.18 0.01ind.106 day 1 L 1), compared with the
rest (lot numbers 3^7:0.29 0.01ind.106 day 1 L 1).
Discussion
The rotifer mass culture protocol during the main
production phase can be compared with other semicontinuous systems. Rotifer density was on the upper
range of reported values, whereas the growth rate
was similar to bibliographical values (Person-Le
Ruyet 1975; Hirata, Yamasaki, Kawagushi & Ogawa
1983; James, Bou-Abbas, Al-Khars, Al-Hinty & Salman 1983; Korstad, Neyts, Danielsen, Overrein & Olsen 1995; Lubzens, Minko¡, Barr & Zmora 1997;
Navarro & Yu¤fera 1998a; Makridis & Olsen 1999).
Maximum culture duration exceeded the majority of
bibliographical values reported in Lubzens et al.
(2001). Similar results were obtained in two studies,
both of which used phytoplankton in either fresh
form with yeast (James et al. 1983) or solely as dry
powder (Navarro & Yu¤fera 1998b). There is one reference (Hirata et al. 1983) that reports a higher culture
duration (appr. 65 days), where phytoplankton and
yeast were used as feed, but rotifer density was kept
at much lower levels (93 30 ind. mL 1). The daily
yield exceeded reported values (James et al. 1983;
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 91–98
Lubzens 1987; Navarro & Yu¤fera 1998b). The e⁄ciency was lower (Navarro & Yu¤fera1998a, b) or similar (Hirata et al. 1983; James et al. 1983) to the
bibliography, depending on the feed; phytoplankton
and yeast showed similar results, whereas phytoplankton in dry form was more e⁄cient. It appears
that the type of feed in£uences food conversion rates
and that phytoplankton should be preferred to dry
feed, where possible. This is in agreement with the
widely accepted view that phytoplankton gives better
results in rotifer culture compared with dry feeds
(Lubzens 1987; Nyonje & Radull 1991; ie, Reitan &
Olsen 1994; Korstad et al.1995; Borowitzka 1997; Lubzens et al. 2001). Overall, the semi-continuous system
of this study produced a high daily yield, for a considerable period of time, using dry feed as food. It is expected that the characteristics of the system could be
improved with a phytoplankton-based diet.
The e⁄ciency of the semi-continuous system can be
further improved through the dilution rate and the
amount of food supplied. For a given dilution rate, the
rotifer population stabilizes at higher densities with increasing food ration (Navarro & Yu¤fera 1998b). In this
study, the quantity of dry feed used per rotifer was lows
er than recommended levels by INVE (M. Vasilakis,
pers. comm.). This allows for a further increase in
the quantity of dry feed used. On the other hand, the
dilution rate is positively related to the growth rate.
At high dilution rates (0.3 day 1), practically all the
food energy is utilized for reproduction giving high
fecundity (Navarro & Yu¤fera 1998a). Thus, if the dilution rate and the feed ration are increased up to a
point, the growth rate and yield of the system could
be further improved. This is one of the advantages of
the semi-continuous system; it can be accordingly
tailored to the needs of the production at any time.
When comparing the two types of culture systems,
the semi-continuous culture showed a signi¢cantly
95
Semi-continuous rotifer mass culture V Kostopoulou et al.
Aquaculture Research, 2012, 43, 91–98
Table 5 Estimated operation cost to produce 500 million rotifers (of approximately weekly duration) in batch vs. semi-continuous mass culture (developed from Suantika et al. 2003)
Batch
Semi-continuous
Item
Description
Quantity
Cost (h)
Quantity
Labour (h)
Electricity (kW)
Water (L)
Feed (kg)
Total running cost
Hours per week
Tank heating/cooling
Pumping of water from drill
s
Selco Sparkle
4.9
7.3
3.330
1.04
73.5
122.6
2.9
5.3
2.350
1.26
51.5
247.6
Cost (h)
43.5
89
62.4
194.9
Minimal cost compared with rest.
longer duration and an almost double daily yield,
compared with the batch, in agreement with the bibliography (Navarro & Yu¤fera 1998a; Dhert et al. 2001;
Lubzens et al. 2001). This can be attributed mostly to
the dilution of the culture medium, resulting in a
better water quality. In the case of the batch system,
the cultures are subjected to highly variable conditions, mainly due to the build-up of metabolic by-products, uneaten food and an unstable microbial
community (Dhert et al. 2001; Conceicao et al. 2010).
The fact that the e⁄ciency did not di¡er between the
two culture systems can be attributed to the amount
of administered food. Higher food usage in the semicontinuous system is probably related to the routine
of daily dilutions of the culture; a fraction of the food
is also lost through the system (Suantika, Dhert,
Sweetman, O’Brien & Sorgeloos 2003). Nevertheless,
the daily management of the semi-continuous system is more e⁄cient in terms of time, e¡ort and water
consumption, compared with the batch system (Table 5). The practical expertise needed is similar in
the two systems, which means that hatcheries can
switch from batch to semi-continuous without
further training of their sta¡.
The residence time of a rotifer population in mass
culture tanks without renewal from stock cultures
had an impact on the daily yield. According to the results, a longer residence time (lot numbers 1, 2) resulted in lower daily yields during the main phase of
production. In other words, time had a negative in£uence on rotifer performance. Di¡erent factors may be
responsible for this, such as dietary, microbiological,
even genetic. A change in diet from phytoplankton
(supplied in stock cultures and upscaling) to dry feed
(mass culture) may adversely a¡ect rotifers. As mentioned above, phytoplankton appears to be of better
quality for rotifers than dry feeds. Therefore, substituting phytoplankton with dry feed during mass culture
96
may have a cumulative negative in£uence on rotifer
performance. Likewise, a shift in bacterial composition
as a result of changing diet (Skjermo & Vadstein 1993;
ie et al.1994) may also in£uence rotifer performance.
A lower daily yield with time could be potentially associated with clonal selection; parthenogenesis leads to a
signi¢cant reduction in clonal diversity with time (Go¤mez & Carvalho 2000). Occasional £uctuations in culture conditions ^ which most of the time go unnoticed
during mass culture production ^ may have a negative
in£uence on a rotifer population composed of few
clones, which have adapted to a narrow set of conditions. In this sense, clonal selection could result in a
reduction in the adaptation potential of a rotifer population with time. Irrespective of the reason, there appears to be a trade-o¡ between rotifer culture
duration and yield, which can be accordingly adjusted
depending on production needs.
The use of the automatic feeder contributed to the
stability of the culture through the frequent addition
of food in small quantities. One of the main problems
in rotifer mass cultures of most hatcheries stem from
the infrequent addition of food in rotifer tanks. As a
consequence, the amount of administered food is,
most of the time, either higher (after the addition of
food) or lower (before addition of next portion) than
required. A sudden increase in the levels of food
added in a tank supports the development of microzooplankton (ciliates) can lead to oxygen depletion
and water quality deterioration. On the other hand,
reduction in food levels can result in underfed rotifers and delays in their development, having direct
consequences on the growth rate (Lubzens et al.
1997; Olsen 2004). In this study, the growth of ciliates
was kept at low levels, compared with the values reported in the literature (Cheng, Aoki, Maeda & Hino
2004). In rotifer mass culture tanks, ciliates mainly
feed on dead rotifers and their faeces (Hagiwara,
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 91–98
Aquaculture Research, 2012, 43, 91–98
Semi-continuous rotifer mass culture V Kostopoulou et al.
Jung, Sato & Hirayama 1995; Cheng et al. 2004; Olsen
2004). Therefore, the low number of ciliates provides
a proof that continuity in feeding helps to reduce ciliate competition, mortality of rotifers and development of necrophagous material. It should be noted
that one of the limitations of the automatic feeder is
related to its capacity; it can only be used with food in
compact form (dry feed, phytoplankton paste).
The estimated cost for rotifer production for the
main parameters of the two culture systems is calculated in Table 5. It is evident that the di¡erence in cost
between the two systems is small. However, when the
semi-continuous system is extended to several weeks,
the abovementioned di¡erence becomes considerable.
Therefore, for a whole production cycle, the semi-continuous system is expected to have a lower running
cost, compared with the batch. Lower costs have been
reported for other semi-continuous as well as continuous and recirculation systems, due to either higher
rotifer total standing stock and/or density among
other factors (Fu et al.1997; Lubzens et al.1997; Suantika et al. 2003). It is therefore suggested that the cost
could be further reduced by increasing the working
rotifer density and/or the culture volume.
Overall, the semi-continuous system as presented
here is more e⁄cient compared with the batch, due to
its higher culture duration, higher daily yield, lower
cost and more e⁄cient daily management. On the
downside, the semi-continuous system is characterized to be a less-e⁄cient food usage. When combined
with the automatic feeder, the semi-continuous system
had a positive in£uence on rotifer mass culture stability. The daily renewal of part (25%) of the culture and
the addition of small quantities of dry food at regular
time intervals (10 min) resulted in a prolongation of
culture duration, reaching a maximum of 41 days.
Further improvement of this system can be achieved
through the manipulation of the dilution rate and food
ration, the type of feed, culture volume and duration.
Therefore, the use of these two systems can potentially
contribute to a more stable and predictable mass production of rotifers in hatcheries.
References
Acknowledgements
The authors would like to thank Dr Maria Jose¤
Carmona (University of Valencia, Spain), Dr Pavlos
Makridis (Institute of Aquaculture, HCMR, Crete,
Greece) and two anonymous reviewers for useful
comments.
© 2011 Blackwell Publishing Ltd, Aquaculture Research, 43, 91–98
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