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Continuous production of Chaetoceros calcitrans in a system suitable forcommercial hatcheries
Heinrich F. Kaspar a,⁎, Elizabeth F. Keys a,1, Nick King a, Kirsty F. Smith a,Aditya Kesarcodi-Watson a, Matthew R. Miller b
a Cawthron Institute, Private Bag 2, Nelson 7042, New Zealandb Marine Products, The New Zealand Institute for Plant & Food Research Limited, 300 Wakefield Quay, Nelson, New Zealand
Chaetoceros calcitrans is a small, fast-growing diatom with a high nutritional value for filter feeders. It is oftenused in hatcheries, particularly in the production of bivalve spat. It is widely produced in batch culture systemsranging from carboys to plastic bags to high volume tanks. Continuous culture of C. calcitrans has generally notbeen successful due to frequent crashes of the culture.We report the continuous culture of C. calcitrans in a hang-ing plastic bag system that is suitable for use in commercial hatcheries. Runs of continuous production lasted upto 125d, with an average bag life of 24.4d. Crashes occurred at irregular intervals in individual bags and causedthe cell concentration to drop by up to three orders of magnitude. Cultures recovered over a few days followinga crash. The temporary reduction of production due to crashes in individual bags was compensated by a30% higher production than required. At dilution rates of 1.0–1.3 volume exchanges d−1 the system produced7–13 × 106 cells mL−1 d−1. The cell diameter mode as determined by a Coulter Counter was 2.8–3.0 μm. Thecells had the typical flat rectangular box shape with well-developed setae. The culturable bacterial populationof the algae culture was 105–107 colony forming unitsmL−1. Genetic integrity of C. calcitrans and monospecificstatus of the continuous culture were maintained over 7 sequential bag-to-bag transfers during a 125-day run.The fatty acid profiles of continuous and batch cultures of C. calcitrans were similar. Growth and survival ofGreenshell™mussel (Perna canaliculus) larvae were identical when fedwith C. calcitrans batch culture or contin-uous culture delivered directly from the harvest line. When continuous culture was first collected over 24 h,diluted and then delivered to the rearing tanks over 24h, larval growth was markedly slower (24d to reach set-tlement competency compared to 21 d for the other two treatments); however the subsequent settlement suc-cess of competent larvae was not statistically different between the three diets (‘Carboy’, ‘Continuous/Harvestline’, ‘Continuous/Feed-out bin’; 33.9–35.5% settled). Concomitant to the differences in larval performance weobserved a difference in larval fatty acid profile: lower-performing larvae had a markedly lower proportion ofmono-unsaturated fatty acids and a markedly higher proportion of poly-unsaturated fatty acids. Lower perfor-mance and different fatty acid profiles of larvae fed the ‘Continuous/Feed-out bin’ diet appeared to be causedby unidentified changes that occurred during storage of the C. calcitrans culture.
Chaetoceros calcitrans is one of the most commonly grown micro-algae in bivalve hatcheries (Tredici et al., 2009). It is used as feed forbrood-stock (Pronker et al., 2008), juveniles (Enright et al., 1986) andlarvae (Rico-Villa et al., 2006) of commercially important shellfishspecies. It has also been used as feed for various other commercially sig-nificant taxa, e.g. echinoderm larvae (Asha andMuthiah, 2006; Carcamoet al., 2005) and brine shrimp (De los Rios, 2001). The species has beenregarded as unsuitable for large-scale production (Muller-Feuga et al.,
4 3 55 2640.aspar).w Zealand.
ghts reserved.
2003b) and it is generally produced in labour-intensive batch culture(Laing, 1979).
Twenty years ago, the cost of algae production represented 20–50%of hatchery operating costs (Coutteau and Sorgeloos, 1992). Secondarysources suggest that algae production is still a major component of thehatchery operating costs (Muller-Feuga et al., 2003b; Sarkis andLovatelli, 2007). Labour has been identified as a major cost componentand continuous culture has been proposed as a potential means to re-duce labour cost (Fulks and Main, 1991; Muller-Feuga et al., 2003a).Continuous algal culture systems, where new medium is constantlyadded to allow passive overflow harvesting of cells at a rate designedto maintain exponential growth, present an expedient and efficientmechanism for bulk algae production. In addition, continuous culturecan be expected to produce algal cells that are more uniform thanbatch-produced cells (potentially leading to more stable and
Table 1Diet regimes for the three treatments compared in a Perna canaliculus larval performanceassay.
Treatment Replicates C. calcitransproduction method
C. calcitrans harvestand delivery method
‘Carboy’a 7 Batch Carboy/feed-out binb
‘Continuous/Feed-outbin’a
7 Continuous Harvest tank/feed-outbinc
‘Continuous/Harvestline’d
6 Continuous Harvest line/dilutionbine
In all treatments, I. aff. galbanawas harvested and delivered to the larvae in the same wayas C. calcitrans in the ‘Continuous/Feed-out bin’ treatment.
a C. calcitrans exclusively in the first 15 d, 10% I. aff. galbana on Day 16, then 20% I. aff.galbana till end of experiment.
b The appropriate amount of culture for a 24 h feed-out period plus 50% surplus wastransferred from the carboy to a 20L feed-out bin anddiluted to 15Lwith 1μmfiltered sea-water. Over the following 24 h, 10 L of this algal suspension was delivered to the headertank in 3mL pulses at 25.9 s intervals.
c A harvest tank collected the continuous production of one bag row for up to 24h. Theappropriate amount of culture for a 24h feed-out period plus 50% surplus was transferredfrom the harvest tank to a 20 L feed-out bin and then diluted and delivered as under (b)above.
d C. calcitrans exclusively in the first 15 d, 10% I. aff. galbana on Days 16–18, then 20% I.aff. galbana till end of experiment.
e The harvest line for the ‘Continuous/Feed-out bin’ treatment first discharged into a1.1 L jug which overflowed into the harvest bin. A process pump delivered 10 L of algaein 3mL pulses at 25.9 s intervals from the jug into a 20-L dilution bin which also received200 L d−1 1 μm filtered seawater over 24 h. The appropriate amount of diluted algal sus-pension was pumped in 3mL pulses to the header tank.
2 H.F. Kaspar et al. / Aquaculture 420–421 (2014) 1–9
predictable larval performance) and the continuous process lends itselfto computer control (Borowitzka, 1997).
Over the past 15 years, the algae production effort at the CawthronInstitute (Nelson, New Zealand) has been divided evenly between upto 2000 L d−1 continuous production of Isochrysis aff. galbana, Pavlovalutheri and Chaetoceros muelleri and up to 140 L d−1 of C. calcitrans in20 L batch cultures. Because C. calcitrans is an excellent feed alga forlarvae of the Greenshell™ mussel Perna canaliculus (Ragg et al., 2010)we have a particular interest in growing it cheaply, reliably and uni-formly. An earlier project (Tannock, 2006) demonstrated the feasibilityin principle of continuous C. calcitrans production in a hanging plasticbag system and estimated that the unit labour cost for continuous pro-duction would be less than one sixth of the unit labour cost for batchproduction.
This report demonstrates the technical feasibility of continuousC. calcitrans production in a system suitable for commercial hatcheries.With emphasis on practicality and relevant parameters, we describethe method and its performance and risks, and we compare the perfor-mance of P. canaliculus larvae fed with C. calcitrans produced in eitherbatch or continuous culture.
2. Materials and methods
2.1. Organisms
C. calcitrans (CCMP 60/00/00 1315) and I. aff. galbana (T-Iso, CCMP1324) were obtained from CSIRO Marine Research, Hobart, Australia.The C. calcitrans strain used in this study is similar to C. calcitransforma pumilum (Robert et al., 2004; Takano, 1968). P. canaliculusbrood-stock was obtained from a commercial farm in the MarlboroughSounds, New Zealand.
2.2. Micro-algae production and harvest
2.2.1. C. calcitrans batch cultureBatch cultures of C. calcitrans were produced in 20 L polyethylene
carboys (Nalgene, Thermo Fisher) according to the routine Cawthronprocedure: autoclaved seawater at 3.5% salinity enriched with Conwaydiatom medium (Sarkis and Lovatelli, 2007); 20–23 °C; continuouslight at 750–1000 lx from cold white fluorescent tubes on one side ofthe carboys (measured using an Advanced Light Meter 840022, SPERScientific Ltd., Scottsdale, AZ); 9.3 Lmin−1 air supplemented with 0.2%CO2; and carboys received 400mL inoculum from a 3 d old 3 L culture(19–29×106 cellsmL−1).
Cultureswere harvested after a 3d incubation near the endof the ex-ponential growth phase, and fed to themussel larvae at the appropriateamounts (see Subsections 2.3.2 and 2.3.3).
2.2.2. I. aff. galbana continuous cultureI. aff. galbana is a major component of our bivalve larval diet (Ragg
et al., 2010). In the present study, the diet consisted of 10–20% of thisspecies after Day 15 of larval rearing (Table 1). The culture was grownin a continuous modified Seasalter system using 40 L bags (clear poly-ethylene; 15cmdiameter; 8 hanging bags per row). Pasteurised naturalseawater (3.5% salinity) was continuously supplemented with stock so-lutions by a processing pump (SMC Pneumatics pump PB1011-01,Indianapolis, USA) to make the Conway medium (Sarkis and Lovatelli,2007) and delivered at 15 L bag−1 d−1 (dilution rate= 0.375 d−1) bya processing pump (SMC Pneumatics pump PA3213-03). The pumpswere controlled by a PLC (programmable logic controller, SchneiderElectric Premium series, Rueil-Malmaison, France). Other conditionswere: 20–23°C; continuous light at 750–850lx from cold white fluores-cent tubes on one side of the bags; and 2.4 Lmin−1 air supplementedwith 0.2% CO2 introduced at the bottom of the bags and exhaustedthrough the outflow. The outflow from the bags was collected in an
aerated bin for 15–24 h before samples were taken for feed-out to thelarvae in the appropriate amounts (see Subsection 2.3.3).
2.2.3. C. calcitrans continuous culture and harvestThe system was similar to that used for I. aff. galbana (Subsection
2.2.2). Bag volumes ranged from 16 L to 29 L. There were 7 hangingbags per row and 1–4 rows. Pasteurised seawater (3.5% salinity) wassupplemented with stock solutions to make the Conwaymediumwith-out silicate (Sarkis and Lovatelli, 2007). This medium was supplied tothe bags at constant rates of 21–43 L bag−1 d−1 (dilution rates rangedbetween 0.6 and 1.6d−1) for several days until steady-state algae pro-duction was documented or the culture failed due to crash or wash-out. Silicate was added separately to each bag (4.725 g d−1 Na2Si2O3 ×5H20 dissolved in 625mL water purified by reverse osmosis, suppliedin 0.4mL pulses to the top of the bags by a PLC-controlled processingpump). Other input conditions were: 20–23 °C; continuous light at750–850 lx from cold white fluorescent tubes on one side of the bags;and 0.9Lmin−1 air supplemented with 1% CO2 introduced in large bub-bles at the bottom of the bags and exhausted through the outflow, thuscreating similar shear forces at the surfaces of all cultures.
Bags were inoculated with 2–5L culture from a batch culture as de-scribed above (Subsection 2.2.1). Medium was allowed to flow into thebags for 1–3 h before inoculation. Aeration and silicate supply werestarted once the bags contained about 5 L culture.
Bags were also inoculated from existing bags. The outflow of a bagwas connected by a silicone hose with a temporary inflow port at thetop of a new bag. The exhaust air pushed the overflow from the existingbag into the new bag. At the same time the new bag received medium,silicate and air. The filling of a new bag took a few hours after which theinoculation hose was removed and the temporary inflow port wasclosed with duct tape.
The cultures were cleaned daily using the following procedures. Inorder to remove fouling from inside the bags they were vigorouslypunched by fist. Fouling at the top of the bags was removed by rubbingthe inside of the bag against itself. Once the fouling was removed fromthe bag sides the flocs were allowed to settle to the bottom of thebags by stopping aeration (5–15min). The accumulated dirt was thenallowed to drain for over 1–3min by removing the air-line. If necessary,the settling/draining process was repeated until most of the dirt was
3H.F. Kaspar et al. / Aquaculture 420–421 (2014) 1–9
removed. Then the air-flow was re-started and the air-line was re-inserted into the bottom of the bag.
The outflow from each bag row was collected in an aerated tank forup to 24h before the required volume was taken for feeding the larvae.Algae were also pumped in 3mL pulses at 30 s intervals from the com-bined outflow of a bag row (harvest line) to a 20L bucket where the cul-turewas continuously diluted twentyfoldwith 1μm filtered seawater asused for larval culture. The diluted algal suspension was continuouslyfed to the larvae in the appropriate amounts (see Subsection 2.3.3).
A simple daily assessment of the culture quality in each bag formedthe basis for the decision to use or discard the product from each bag forthe following 24h. Cultures that showed significant overnight accumu-lation of ‘fur’ on the bags and/or flocculation and/or significantly lightercolour than at the previous assessment (visual signs of a ‘crash’) weredisconnected from the harvest line.
Each bag could be individually switched between continuous modeand batch mode. This was achieved by turning on/off the supply ofmedium and silicate. Crashed cultures were often run in batch modefor 1–4d to allow the recovery of the cell density.
A bag was used until it could not be cleaned any more or its culturecrashed to a cell concentration that would have required many days ofrecovery, as indicated by complete loss of colour or b104 cells mL−1.Media supply lines and harvest lines were steam-cleaned at 6 weekintervals.
2.3. Larvae
2.3.1. Spawning, fertilisation and incubationP. canaliculus brood-stock was spawned by temperature shock
(16±5 °C). Males and females were spawned separately and gameteswere stored at 4 °C for up to 4h. The eggs were pooled and transferredto 20 L seawater at 16 °C. Sperm was pooled and then added in smallquantities at 5min intervals to the eggs while they were gently stirreduntil several sperm were visibly attached to each egg (bright field mi-croscopy, ×200). About 60million fertilised eggs were transferred to atank containing 5000 L gently aerated seawater at 16 °C. After 24h thetemperature was raised gradually over 15h to 19.5°C. After a total incu-bation time of 42 h the D-stage larvae were harvested by draining thetank contents through a 45 μm screen. The larvae were rinsed and re-suspended in 5 L seawater (20 °C) for counting and dispensing intothe larval rearing tanks.
2.3.2. Rearing system and methodSeawater (1μm filtered, 19°C) was supplied continuously into three
header tanks, each tank representing an experimental treatment(C. calcitrans culture×feed outmethod, see Subsection 2.3.3). Algae cul-ture was supplied to the header tanks by PLC-controlled processingpumps (SMC Pneumatics pump PB1011, Indianapolis, USA; Festo Pneu-matics clear polyurethane tubing, 6mmØ, Esslingen, Germany) to eachheader tank. From here, the water was supplied to the larval rearingtanks by gravity through manifolds.
The larvae were reared in small flow-through tanks as describedin detail by Ragg et al. (2010). Briefly, the system consisted of 2.4 Lbullet-shaped acrylic tanks with a 60 mL min−1 continuouswater + food supply through a glass tube and an outflow through a45μmpolyethylene screen. The containerswere aerated through anoth-er glass tube at 0.5Lmin−1 and arranged in a double row on both sidesof a drain collecting the overflows. Tanks receiving the same treatmentwere spread evenly over the length and sides of the drain. The watertemperature in the rearing tanks was 20.0–20.6 °C.
Outflow screens were cleaned daily with a hot water spray. Larvalrearing tanks were cleaned every second or third day with sodium hy-pochlorite, hot water and brush. Header tanks, algae supply lines andwater supply lines/manifolds were cleaned as required every 3–5 dwith sodium hypochlorite and/or hot water.
Larvae were dispensed into the rearing tanks to give a starting den-sity of 208 larvae mL−1. At 2–3 day intervals the tanks were drainedthrough a 45μmmesh screen and the larvaewere cleanedwith a gentleseawater spray. After re-suspension of the larvae in 800mL seawater,samples were transferred into 4 mL tissue culture dish wells forcounting (3 × 0.1 mL, see Subsection 2.4.3), size measurements(1×1mL, see Subsection 2.4.3) and fatty acid analysis (see Subsection2.4.4).
The algal concentration in the larval rearing tanks was maintainedbetween 7000 and 30,000 cellsmL−1. The appropriate number of algalcells to feed out at each 24-hour interval was based on algal consump-tion during the previous 24-hour interval.
On Day 19 post fertilisation the larvae were screened on an 85 μmmesh screen. Very few larvae passed through this screen; they werediscarded. The 45 μm mesh screens in the tanks were replaced with75 μm mesh screens.
2.3.3. Experimental designThe aim of this experiment was to compare larval performance
(growth, survival, settlement competency, settlement success) whenfed with C. calcitrans produced in either batch or continuous culture.Table 1 shows the diet regimes for the three treatments tested.
On Day 21 (‘Carboy’ and ‘Continuous/Harvest line’ treatments) orDay 24 (‘Continuous/Feed-out bin’ treatment) the competent larvaewere retained on a 178 μm screen and the smaller larvae werediscarded. A known number of competent larvae (usually 5000) werereturned to the larval rearing tanks, together with a 1 m piece of coir(thin coconut fibre rope). The larvaewere allowed to settle over the fol-lowing 8d, and afterwhich the coir pieceswere frozen for later determi-nation of the number of larvae that had settled and metamorphosedsuccessfully.
2.4. Analyses
2.4.1. Algae bagsThe culture volume of each bag was individually determined at the
end of the bag's life by draining the culture into graduated containers.The individual bag flow was determined by collecting the outflow ofeach bag in a 1 L measuring cylinder over a suitable time interval (10–20min). The dilution rate of each bag was calculated individually fromits volume and flow rate. Volumetric production rates were calculatedwhere a culture was in steady state for at least 4 d (cell concentrationvaried by b20%).
2.4.2. AlgaeCulture samples were collected from individual bags by removing
the air line from the bottom of the bag and draining 200–300mL cultureinto a beaker. This sample was immediately used for measurements oftemperature and pH. A 0.1 mL aliquot was then removed for a serial10-fold dilution in autoclaved seawater and counting of colony formingunits. The dilutions were plated (0.1 mL; triplicates) on CASO agar(Merck) made to 2% salinity with natural seawater and MilliQ water.The plates were incubated at 22 °C, and the bacterial colonies werecounted after 3 d. A further 0.1 mL aliquot was used to measure cellsizes and numbers by a Coulter Counter (MS4, Beckman-Coulter, Fuller-ton, CA; the standard deviation for 10 replicate samples was b8% andb4% of the mean for particle count and mode cell diameter,respectively).
The algal density in the larval rearing tanks was measured with ahand-held fluorometer (Cyclops-7, Turner Designs, Sunnyvale, CA).The fluorometer readings were translated into cell concentrationsbased on the linear relationships established between the fluorometerreadings and the Coulter Counter readings.
Culture samples were pelleted for DNA sequencing by gentle centri-fugation (10mL; 542×g, 15min, RT). Genomic DNAwas extracted fromthe resultant pellet using i-genomic CTB DNA extraction mini kits
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Fig. 1. A. Typical time course of C. calcitrans cell concentration in the first 12 d of continu-ous culture using hanging bags. The bags were inoculated on Day-1 immediately aftersteam-cleaning of themediumsupply system, and started overflowing onDay 0. B. Typicalbacterial counts (Colony Forming Units, CFU) in the same continuous C. calcitrans culturesand in the incoming medium over the same period. Error bars represent 95% confidenceintervals of triplicate measurements.
4 H.F. Kaspar et al. / Aquaculture 420–421 (2014) 1–9
(Intron, Gyeonggi-do, South Korea). The internal transcribed spacer(ITS) region from the 3′-end of a small subunit rDNA (SSU) to the 5′-end of a large subunit rDNA was amplified using the universal primersITS1 (modified from Gottschling et al. (2005)) and ITS4 (White et al.,1990). An approximately 1.7 kb region of the SSU rDNA was amplifiedusing the eukaryote-specific primers EukA and EukB (Medlin et al.,1988). PCR products were purified with an AxyPrep™ PCR clean-upkit (Axygen Biosciences, CA). An external contractor (Waikato Universi-ty DNA Sequencing Facility, Hamilton, New Zealand) sequenced the ITSregion in both directions using the PCR primers and the SSU region inone direction using the EukB primer. Sequence chromatograms wereexamined visually and any base-calling errors were corrected manuallyusing the BioEdit Sequence Alignment Editor (Hall, 1999). The se-quences were compared to existing sequences in GenBank using theBLAST online software (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Images of live algal cells were produced with an Olympus BX-51compound microscope using a U Plan FL N oil 100× NA1.30 objective,differential interference phase contrast (DIC) and Olympus analySIS LSResearch Five software v2.4.
2.4.3. LarvaeLarvae taken for counting and sizemeasurements (Subsection 2.3.2)
were anaesthetised with ethanol. The larvae were counted under anOlympus CK2 inverted microscope at ×40 magnification. Larvae takenfor sizemeasurementwere photographed (Olympus C7070 digital cam-era mounted on an Olympus CK2 inverted microscope, ×40). The im-ages were analysed using ImageJ software (version 1.40g; http://rsb.info.nih.gov/). A customised particle recognition macro was used toisolate individual larvae and determine Feret's diameter (the longestmeasurement across an ovoid) as a measure of shell length. To countthe larvae that had successfully settled and metamorphosed at theend of the trial, the coir samples were thawed and then rinsed with astrongwater jet onto a 200μm screen. The post-larvae were transferredinto a beaker and suspended in 500mL water. Quadruplicate samples(10 mL) were then counted in a Petri dish under an Olympus CK2inverted microscope at ×40 magnification. Only individuals that hadclearly grown beyond the pediveliger stage were counted.
2.4.4. Fatty acidsAlgae samples were taken from carboys and harvest tanks at the
time of transfer to feed-out bins, and at the same time from the harvestline. Algal culture (100 mL) or 50,000 larvae (aliquot of the 800 mLconcentrate from Subsection 2.3.2) were filtered onto a glass microfibrefilter (LBS0GFF, pore size 1 μm, Labserv Auckland). The filters were im-mediately folded, wrapped in aluminium foil and frozen at−80 °C.
Lipids were extracted from the filters via a modification of Matyashet al. (2008). Methanol (1.5mL) was added to an about 200mg sample,which was placed into a glass tube with a Teflon cap and vortexed for1 min. Methyl tertiary butyl ether (MTBE, 5 mL) was added and themixture was shaken for 1 h at room temperature. Mass spectroscopy(MS)-grade water (1.25mL) was added for phase separation. The sam-plewas vortexed again (1min) and centrifuged at 1300×g for 5min. Theupper (organic) phase was collected, and the lower phase was re-extractedwith 2mLMTBE/methanol/water (10:3:2.5, v/v/v). Combinedorganic phases were dried in a rotary evaporator. A small amount ofmethanol was added to the organic phase during rotary evaporationto speed up the process. Extracted lipids were dissolved in 1 mL ofchloroform/methanol (4:1, v/v) for storage.
Gas chromatography of fatty acidmethyl esters (FAMEs): A subsam-ple (5–10mg) of the extracted oil was trans-methylated in methanol/chloroform/hydrochloric acid (10:1:1, by volume) for 1 h at 100 °C.After addition ofwater, themixturewas extracted three timeswithhex-ane:chloroform (4:1, v/v) to obtain FAMEs, which were then concen-trated under nitrogen. Samples were made up to a known volumewith an internal injection standard (19:0 FAME NuChek, Elysian, MN,USA). Samples were analysed by gas chromatography (GC) using a
Shimadzu 2010 GC instrument equipped with a Restek GTx silica capil-lary column (30 m × 0.25 mm i.d., 0.25 μm film thickness), and massspectroscopy (MS) on a Shimadzu 2010 QP GC–MS instrument. FAMEsamples were injected at an oven temperature of 60 °C, which was in-creased to 100 °C at a rate of 40 °C min−1, then to 170 °C at a rate of10 °Cmin−1. The temperature conditions were further adjusted as fol-lows: increase at 5 °C min−1 to 185 °C, hold for 2 min, increase at3 °C min−1 to 197 °C, increase at 0.5 °C min−1 to 199 °C, hold for1 min, increase at 5 °C min−1 to 230 °C, hold for 3 min, increase at5 °Cmin−1to 250°C, and hold for 5min. Heliumwas used as the carriergas.
2.4.5. Data analysisFatty acid and larval performance data for the different treatments
were compared using ANOVA (pb0.05). Specific treatment differenceswere identified by the Tukey HSD post-hoc test. Where data did notmeet ANOVA assumptions, treatments were compared using the non-parametric Kruskal–Wallis test (p b 0.05) with post-hoc analysis doneby 2-tailed multiple comparisons of p values. All analyses were per-formed using STATISTICA 8.0 (Stat Soft Inc.).
3. Results
3.1. Continuous C. calcitrans production
Between January 2010 and July 2012 we carried out 13 runs of con-tinuous C. calcitrans production. The duration of these runs ranged from5 to125d,with an average of 42d. Run11was typical (Fig. 1A): Thebags
Fig. 3.Mode cell diameter of C. calcitrans in seven bags determined daily over thefirst 28dof continuous culture. The value for Day 0 was measured in the inoculum (batch culture).
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started overflowing at a cell concentration of about 5 × 106 cells mL−1
(Day 0). The cultures grew to 107 cells mL−1 (Day 4) before crashingto 105–106 cells mL−1 over the following 3–4 d. The reduction in celldensity was accompanied by the appearance of suspended flocs and/or ‘fur’ on the bag side, as well as yellow foam in the harvest tank. Thecultures were cleaned daily over the following days, while the culturedensity recovered. A similar crash/recovery cycle often occurred be-tween Day 13 and Day 22, before the cultures settled into a longer peri-od of stable production with densities around 107 cells mL−1. Somecrashes led to a thousand-fold reduction of the cell concentrationfollowed by slow recovery, and twenty-day periods of steady cell con-centration were common in later runs (results not shown).
3.2. Bacterial counts
Culture medium and micro-algae cultures were not axenic (seeFig. 1B for an example). Immediately after steam cleaning of themediumsupply system, the bacterial count of the medium entering the culturebags (measured as colony forming units, CFU) was low but the mediumwas not sterile. The bacterial count of the inoculum was also low, givingan initial bacterial count in the bags of about 104CFUmL−1.Within a dayof continuous culture, the bacterial count reached 106 to 107 CFUmL−1
and remained at that level until it started to decline in the second halfof the algal crash (Days 5–6). This decline continued during the recoveryof the algal population and the subsequent period of constant algal celldensity. At the termination of the experiment the bacterial count of thealgal cultures (3.7 × 104–6.4 × 105 CFU mL−1) was no more than anorder of magnitude higher than the bacterial count in the medium(3.3×104CFUmL−1).
3.3. Production rate
Steady state conditions were obtained at dilution rates of 0.72–1.43 d−1 (Fig. 2). At lower dilution rates the cultures tended to be lessstable, and at dilution rates above 1.43 d−1 wash-out was observed.Most steady state conditions were obtained at dilution rates of 1.0–1.3 d−1 and the corresponding volumetric production rates were 7–13×106 cellsmL−1 d−1 or 0.15–0.23mm3biovolumemL−1 d−1.
3.4. Cell size and morphology
The mode cell diameter of the inocula (2–3 day old batch cultures)was 3.7–3.9μm. This dimension decreased over the first 2weeks in con-tinuous culture and levelled off at 2.8–3.0 μm (Fig. 3). The cells from
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batch cultures appeared near spherical while the cells in continuous cul-ture were typically rectangular with long setae (Fig. 4).
3.5. Bag-to-bag transfer
Run 12was started on 13 July 2011with 8 bags and one carboy of in-oculum. The full complement of 14 bags was obtained by bag-to-bagtransfer between Days 11 and 24 of the run. The run was terminatedon 15 November after 67 bags had been used, all inoculated fromexisting bags. Not considering the 14 bags that were hanging on theday of experiment termination, the average life time of the bags was24.4d.
The amplified sequences of all algal samples taken throughout therun were identical and homologous with C. calcitrans (ITS: GenBankaccession number DQ358117; SSU: GenBank accession numberEU240880; Fig. 5).
3.6. Fatty acid profiles
Algae fed to the larvae were sampled on Days 8, 12, 16 and 21(C. calcitrans) and Days 16 and 21 (I. aff. galbana) of the larval rearingperiod. The C. calcitrans fatty acid composition did not change signifi-cantly between the sampling dates (p N 0.05; data not shown). Therewere however small but significant differences in the C. calcitrans fatty
Fig. 4. Photomicrographs (differential interference contrast, ×400) of C. calcitrans cellsgrown in batch culture (A) and continuous culture (B). Scale bar=20 μm.
Fig. 5. Continuous culture of C. calcitrans using bag-to-bag transfer, July–November 2012.Horizontal bars represent the lifetime of individual culture bags. Bags were inoculated bybag-to-bag transfer from an existing bag as indicated by vertical arrows and correspond-ing shading. √ = Culture samples taken for genotyping and culture identified asC. calcitrans.
100
120
140
160
180
200
220
240
260
280
5 10 15 20 25
She
ll le
ngth
(µm
)
Larval age (d)
Carboy
Continuous/Feed-out bin
Continuous/Harvest line
C. calcitrans culture and delivery method:
Fig. 6. Shell growth of P. canaliculus larvae reared in small flow-through tanks on a dietconsisting largely of C. calcitrans that was produced and delivered by three differentmethods. Larvae receiving the ‘Carboy’ and ‘Continuous/Harvest line’ diets were settledon Day 21, and larvae receiving the ‘Continuous/Feed-out bin’ diet were settled on Day24. Error bars represent 95% confidence intervals, 6–7 replicates.
6 H.F. Kaspar et al. / Aquaculture 420–421 (2014) 1–9
acid composition between the different production/delivery treat-ments. The fatty acid composition of I. aff. galbanawas markedly differ-ent from that of C. calcitrans (lower MUFA and EPA, higher PUFA andDHA; Table 2).
3.7. Larval performance
Larvae receiving the ‘Continuous/Feed-out bin’ treatment grewslower than larvae receiving the other two treatments (Fig. 6). Thisslower growth required a 3d longer larval rearing period before thema-jority of the larvae were ready to settle. The larvae concentration de-creased more or less linearly over the rearing period and there was nosignificant difference between the three treatments (Fig. 7). The ex-tended larval rearing period for the ‘Continuous/Feed-out bin’ treat-ment resulted in fewer competent larvae than the other two treatments.
Table 2Fatty acid composition (mean± 95% confidence intervals) for three different methods ofproduction and delivery of C. calcitrans (CC, 4 samples) and continuously produced I. aff.galbana (2 samples). Values in a row not sharing a superscript are statistically different(p b 0.05). No statistical differences were detected for palmitic acid. SFA = saturatedfatty acids, MUFA = mono-unsaturated fatty acids, EPA = eicosapentaenoic acid,DHA=docosahexaenoic acid, PUFA=poly-unsaturated fatty acids.
Larvae from the ‘Carboy’ and ‘Continuous/Harvest line’ treatmentswere allowed to settle on Day 21 when they had reached an averageshell length of 235 and 250 μm, respectively. On that day, larvae fedwith the ‘Continuous/Feed-out bin’ diet measured 208 μm and did notshow pediveliger characteristics. They were settled on Day 24 whentheymeasured 221μm.On the day chosen for settlement the proportionof competent larvae (retained on a 178μmscreen)wasmarkedly higherfor the ‘Continuous/Harvest line’ treatment than for the other treat-ments. Therewas no significant difference in the subsequent settlementpercentage between the three treatments (Table 3).
On the last day of larval rearing the larvae in all treatments hadsimilar proportions of saturated fatty acids but the proportions of unsat-urated fatty acids were markedly different between ‘Continuous/Feed-out bin’ and the other treatments (Table 4). The fatty acid profileswere most similar between ‘Carboy’ and ‘Continuous/Harvest line’(insignificant differences in 19 of 21 comparisons) and least similar be-tween ‘Continuous/Feed-out bin’ and the other treatments (insignifi-cant differences in 3–4 of 21 comparisons).
0
50
100
150
200
250
Larv
ae C
once
ntra
tion
(larv
ae m
L-1)
Larval age (d)
Carboy
Continuous/Feed-out bin
Continuous/Harvest line
C. calcitrans culture and delivery method:
5 10 15 20 25
Fig. 7. Net survival represented by concentration of P. canaliculus larvae reared in smallflow-through tanks on a diet consisting largely of C. calcitrans that was produced and de-livered by three different methods. Larvae receiving the ‘Carboy’ and ‘Continuous/Harvestline’ diets were settled on Day 21, and larvae receiving the ‘Continuous/Feed-out bin’ dietwere settled on Day 24. Error bars represent 95% confidence intervals, 6–7 replicates.
Table 3P. canaliculus larvae at settlement (mean± 95% confidence intervals, 6–7 replicates) after rearing on a diet consisting largely of C. calcitrans that was produced and delivered by threedifferent methods. Values in a column not sharing a superscript are statistically different (p b 0.05). No statistical differences were detected for the percentage of competent larvae thatmetamorphosed successfully.
Production/delivery method
Age at settlement(d)
Shell length atsettlement (μm)
Larvae concentration atsettlement (larvae mL−1)
Proportion retained on 178 μmscreen at settlement (%)
% of competent larvae thatmetamorphosed successfully
7H.F. Kaspar et al. / Aquaculture 420–421 (2014) 1–9
4. Discussion
The data presented here demonstrate that C. calcitrans can be pro-duced continuously in simple photobioreactors at commercial hatcheryscale. At dilution rates of 1.0–1.3 d−1 the system produced 7–13 × 106 cells mL−1 d−1 over several months. Continuous C. calcitransculture over a commercially useful period (30 d) has been achieved ina small laboratory-scale flat-panel photobioreactor (Kutako andPowtongsook, 2006); however the steady-state conditions were notwell defined in terms of cell concentration and the highest volumetricproduction rate (1.32 × 106 cells mL−1 d−1) was lower than those ob-tained in this study. Semi-continuous C. calcitrans production in a 17 Llaboratory scale airlift photobioreactor (ALPBR) gave a volumetric pro-duction rate (9.45 × 106 cells mL−1 d−1) similar to those presentedhere (Krichnavaruk et al., 2005).
To our knowledge, this is the first description of C. calcitrans contin-uous culture in a system suitable for commercial hatcheries. Continuousproduction was achieved in an open, non-sterile system without con-tamination by other algae. Although crashes did occur the system wasreliable due to the possibility of quickly starting new production units(bags) by bag-to-bag transfer. The same procedure also rendered thesystem easily scalable: more bags could be brought into production orthe number of bags could be reduced at short notice so that productioneffort and expense were easily adjusted to the requirement. Allcomponents of the system were readily available and the productionof heat-sealed polyethylene bags was the only specialist skill requiredfor the maintenance of the system. Key process parameters were con-trolled independently (temperature, light, aeration, CO2 flow) or by a
Table 4Fatty acid composition (mean±95% confidence intervals, 6–7 replicates) of P. canaliculuslarvae on the last day of rearing in small flow-through tanks on a diet consisting largely ofC. calcitrans that was produced and delivered by three different methods. Values in a rownot sharing a superscript are statistically different (p b 0.05). SFA= saturated fatty acids,MUFA = mono-unsaturated fatty acids, EPA = eicosapentaenoic acid, DHA =docosahexaenoic acid, PUFA=poly-unsaturated fatty acids.
g/100 g oil
Carboy Continuous/Feed-out bin Continuous/Harvest line
14:00 6.6± 1.6b 4.2± 0.1c 7.5± 0.3a
16:00 18.0± 1.0c 20.0± 0.6a 18.6± 0.3bc
18:00 5.2± 0.8b 6.7± 0.1a 4.8± 0.2b
∑SFA 32.2± 1.8b 33.5± 1.0a 33.5± 1.2a
16:1(n-7) 18.8± 3.7a 6.2± 0.1b 17.1± 0.3a
18:1(n-7) 5.2± 0.8a 4.6± 0.3b 5.4± 0.4a
18:1(n-9) 1.4± 0.5a 1.3± 0.2a 1.2± 0.1a
20:1(n-9) 1.7± 0.6b 4.6± 2.7a 1.4± 0.2b
20:1(n-11) 1.9± 0.2b 1.6± 0.1c 1.8± 0.1b
∑MUFA 29.5± 3.9a 18.6± 3.0b 27.5± 0.5a
18:3(n-3) 1.4± 0.3a 1.2± 0.1a 1.2± 0.3a
18:4(n-3) 1.6± 1.9ab 0.8± 0.1b 2.5± 0.1a
20:4(n-3) 2.2± 0.3c 3.2± 0.2a 2.1± 0.0c
20:5(n-3) (EPA) 17.6± 4.0b 14.3± 0.7c 19.0± 1.0b
22:4(n-3) 1.9± 0.3b 2.6± 0.2a 1.8± 0.1b
22:6(n-3) (DHA) 5.2± 0.9b 14.1± 0.7a 4.8± 0.1bc
∑(n-3) 29.9± 4.1b 36.1± 1.5a 31.4± 0.9b
16:3(n-4) 2.6± 1.0a 0.7± 0.0b 3.1± 0.1a
18:2(n-6) 2.3± 1.7ab 2.6± 0.2a 1.5± 0.1bc
22:4(n-6) 0.3± 0.1b 1.9± 0.2a 0.3± 0.1b
∑PUFA 38.2± 2.3b 47.7± 2.1a 38.9± 0.7b
PLC (water flow, nutrient flow, silicate flow). The PLC also controlledthe delivery of continuously harvested algae to the larval rearingtanks. The system addressed most key considerations for hatcheryalgae production (Borowitzka, 1997; Fulks and Main, 1991) in a simpleway and was successfully used for the production of an important andtemperamental species.
The design and operation parameters for this study were similar tothose used for the continuous culture of I. aff. galbana, P. lutheri andC. muelleri at the Cawthron hatchery, and to the parameters for optimaleicosapentaenoic acid production by Phaeodactylum tricornutum in avertical column photobioreactor (Miron et al., 1999). With a maximumlight path of 15cm and cell densities commonly around 107 cellsmL−1
the cultures were probably light-limited. A flat plate reactor with avery short light path (e.g. 5–10 mm) would remove light limitation(Richmond, 2004) however this type of reactor is difficult to keepclean and the operation of many flat plate modules to allow for rapidscaling and certainty of quality and quantity of algal supply would bemore complex than the operation of the same total reactor volume indisposable tubular reactors as used in this study. Based on a work byTannock (2006), shear stresswasminimisedwith a relatively large noz-zle diameter and a minimal aeration rate (Barbosa et al., 2004; Michelset al., 2010). Loubiere et al. (2009) addressed the problem of biofoulingwith a swirling flow through a rigid reactor tube, while in this study anacceptable average bag life of 24.4 d was achieved by rubbing andtapping to mechanically remove fouling. Although there is convergencetowards practically achievable compromises between productivity,simplicity, reliability and cost effectivity, we concur with Carvalhoet al. (2006) that ‘there is no such thing as the best reactor system’
and ‘the most suitable system is situation-dependent’.At least three systems for the continuous culture of micro-algae in
hatcheries are commercially available: (1) the reactor developed byLoubiere et al. (2009) and produced by Jouin Solutions Plastiques(http://www.jouin.com/; accessed 07/06/2013); (2) the CAPS systemproduced by Seasalter Shellfish (http://www.seasaltershellfish.co.uk/;accessed 07/06/2013); and (3) the Biofence photobioreactor marketedby Varicon (http://www.variconaqua.com/index.html; accessed 07/06/3013). To our knowledge, none of these reactors has been used suc-cessfully for the continuous culture of C. calcitrans.
Continuous cultures that had been developed with inoculum fromcarboys (batch culture) generally crashed a few days after the start ofthe treatment. This observation has also been made in other laborato-ries (e.g. Mike Williams, Victorian Shellfish Hatchery Queenscliff,Australia, pers. comm.); however the underlying reasons for this phe-nomenon have yet to be established. The life cycle of diatoms (FrenchandHargraves, 1985) indicates that, given a high degree of synchronisa-tion within a culture, the mass release of spermatogonia might lead tothe concomitant release of cytoplasm and other cell debris capable ofadhering to cells and thus forming flocs that represent a large portionof the culture's biomass. Further crashes of varyingmagnitude occurredthroughout the lifetime of the bags at unpredictable intervals and led totemporary reductions of algae production until the cultures had recov-ered. Crashing cultures formed flocs and/or settled like a fur on thebag sides. These aggregations were easy to remove from the bags, andthe remaining culture consisted largely of individual cells that formedthe foundation for the recovery. Ageing and nutrient-limitedC. calcitrans cultures are known for their propensity to release
8 H.F. Kaspar et al. / Aquaculture 420–421 (2014) 1–9
carbohydrate polymers (Corzo et al., 2000), possibly rendering themsticky. In this study, cells of the crashing cultures were young (mediancell age 0.7–1.4 d) and the medium and culture conditions were de-signed for light limitation. Exopolymer productionmay be the responseto a wider range of stressors than described to date, or it may be a nat-ural physiological phenomenon occurring in all phases of growth as de-scribed for Chaetoceros affinis (Myklestad et al., 1989). Crash-relatedalgal shortages were avoided by producing 30% more algae thanrequired.
The cell size decreased markedly from carboy inoculum to continu-ous culture. In C. calcitrans batch culture, silica limitation leads to in-creased cell size (Laing, 1985). Similarly, Chaetoceros sp. ‘minus’ cellsgrew larger over time in batch culture (Robert et al., 2004). The reversesize trend described here in continuous culture may be due to the re-moval of nutrient limitation after bag inoculation. The shape of thecells from our continuous culture was similar to that observed byRobert et al. (2004) in batch culture.When he limited the silicate supplyto continuous cultures, Tannock (2006) observed distended and sphe-roidal C. calcitrans cells similar to those in carboys used for bag inocula-tion in this study. Nutrient availability appears to be more importantthan culture system/process in determining cell size and shape.
The system used for continuous C. calcitrans production was open tothe outside atmosphere. Also, the growth mediumwas pasteurised butnot sterile, and the inocula from carboys often contained a small bacte-rial population. Axenic production was not possible and not intended.Although the bacterial population in the continuous culture initiallyreached 107 CFUmL−1, it then decreased to levels only slightly abovethe CFU count of the medium. The final bacterial density waslower than earlier measured in continuous I. aff. galbana bag culture(106–107 CFU mL−1, A. Kesarcodi-Watson, unpublished data) andC. calcitrans carboys (Ragg et al., 2010). In our larval rearing experimentthe algal cultureswere diluted 50–350fold between culture bag and lar-val rearing tank,whichmeans that the contribution of CFUs to the larvalrearing tanks was similar between algal culture and seawater (typically103 CFU mL−1; A. Kesarcodi-Watson, unpublished data). The bacterialloading of the continuous C. calcitrans culture presents a risk for the bi-valve larvae similar to that of other algal cultures produced in an opencontinuous system.
Minor differences in the fatty acid composition of C. calcitrans wererecorded between batch and continuous culture. There was no signifi-cant difference in the fatty acid composition between continuouslygrown cells that were collected over 24h in the harvest bin and batch-produced cells. Previous time-series studies of our C. calcitrans batchculture confirmed the general lipid and fatty acid compositions and itsstability over several days, with a few marked exceptions in the earlylogarithmic phase when low cell densities preclude the harvest of abatch culture for commercial feeding (Miller et al., 2012; Ragg et al.,2010). In terms of fatty acid composition there was no marked differ-ence between the two culturing methods.
The storage of continuously produced C. calcitrans in harvest tankand feed-out bin had a negative effect on larval performance and conse-quently spat yield. A range of physical, chemical and/or biologicalchanges during storage of the algae may have had a negative effect onlarval performance, and further work is required to identify the natureof such change(s). In this context it is worth noting that storage of con-tinuously produced C. calcitrans in the harvest tank for up to 24h had lit-tle effect on its fatty acid composition (Table 2), and substantial changesin the fatty acid composition of continuously produced culture duringfeed-out were unlikely considering the time-series data published byMiller et al. (2012) and Ragg et al. (2010).
The fatty acid profile of the feed algae was not obviously reflected inthe fatty acid profile of the larvae, indicating that the larvae were capa-ble of modifying the fatty acids ingested with an almost exclusiveC. calcitrans diet from both, batch and continuous cultures, to suit theirneeds. The reason for the differences in larval fatty acid profiles maytherefore be related to the unidentified causes of different larval
performance rather than to the fatty acid profiles of the diets. The datasuggest that continuously produced C. calcitrans is best fed immediatelyafter harvest as done in the ‘Continuous/Harvest line’ treatment.
C. calcitrans alone or in combination with other microalgal species isa good diet for filter feeder larvae and will adapt to a wide range ofculturing conditions (Anning et al., 2001; Banerjee et al., 2011;Raghavan et al., 2012). This study demonstrates the technical feasibilityof continuous C. calcitrans production in a system suitable for commer-cial hatcheries. Since the conclusion of the experiments reported here,continuously produced C. calcitrans has been used as the major dietcomponent in the production of 4 commercial batches of Pacific oyster(Crassostrea gigas) spat (83, 61, 60 and 52 million eyed larvae; A.Janke, pers. comm.), and a pilot scale batch of P. canaliculus larvae hasbeen reared successfully with continuously produced C. calcitrans assole food (Kaspar et al., unpublished data).
Acknowledgements
We thank Simon Tannock and Johan Quist for demonstrating thatcontinuous culture of C. calcitrans is biologically feasible in a simplephotobioreactor. Cara McGregor, Nicky Roughton, Jolene Taylor, Jona-thanMorrish, AshleighWatts andMike Packer provided excellent tech-nical assistance, andNormanRagg gave valuable constructive critique ofthe manuscript. This work was funded by the New Zealand Govern-ment's Ministry of Business, Innovation, and Employment, contractCAWX0802: ‘Adding Value to New Zealand's Cultured Shellfish Indus-try: Maximising Profit, Minimising Risk’.
References
Anning, T., Harris, G., Geider, R.J., 2001. Thermal acclimation in the marine diatomChaetoceros calcitrans (Bacillariophyceae). Eur. J. Phycol. 36, 233–241.
Asha, P.S., Muthiah, P., 2006. Effects of single and combined microalgae on larval growth,development and survival of the commercial sea cucumberHolothuria spinifera Theel.Aquac. Res. 37, 113–118.
Banerjee, S., Hew, W.E., Khatoon, H., Shariff, M., Yusoff, F.M., 2011. Growth and proximatecomposition of tropical marine Chaetoceros calcitrans and Nannochloropsis oculatacultured outdoors and under laboratory conditions. Afr. J. Biotechnol. 10, 1375–1383.
Barbosa, M.J., Hadiyanto, Wijffels, R.H., 2004. Overcoming shear stress of microalgae cul-tures in sparged photobioreactors. Biotechnol. Bioeng. 85, 78–85.
Borowitzka, M.A., 1997.Microalgae for aquaculture: opportunities and constraints. J. Appl.Phycol. 9, 393–401.
Carcamo, R., Candia, A.I., Chaparro, O.R., 2005. Larval development and metamorphosis inthe sea urchin Loxechinus albus (Echinodermata: Echinoidea): effects of diet type andfeeding frequency. Aquaculture 249, 375–386.
Carvalho, A.P., Meireles, L.A., Malcata, F.X., 2006. Microalgal reactors: a review of enclosedsystem designs and performances. Biotechnol. Prog. 22, 1490–1506.
Corzo, A., Morillo, J.A., Rodriguez, S., 2000. Production of transparent exopolymer particles(TEP) in cultures of Chaetoceros calcitrans under nitrogen limitation. Aquat. Microb.Ecol. 23, 63–72.
Coutteau, P., Sorgeloos, P., 1992. The use of algal substitutes and the requirement for livealgae in the hatchery and nursery rearing of bivalve molluscs: an international sur-vey. J. Shellfish Res. 11, 467–476.
De los Rios, P., 2001. Growth of Artemia franciscana and A. persimilis (Crustacea,Anostraca) under controlled conditions. Rev. Biol. Trop. 49, 629–634.
Enright, C.T., Newkirk, G.F., Craigie, J.S., Castell, J.D., 1986. Evaluation of phytoplankton asdiets for juvenile Ostrea edulis L. J. Exp. Mar. Biol. Ecol. 96, 1013.
French, F.W., Hargraves, P.E., 1985. Spore formation in the life cycles of the diatomsChaetoceros diadema and Leptocylindrus danicus. J. Phycol. 21, 477–483.
Fulks, W., Main, K.L., 1991. The design and operation of commercial-scale live feeds pro-duction systems. In: Fulks, W., Main, K.L. (Eds.), Rotifer and Microalgae Culture Sys-tems. The Oceanic Institute, Honolulu, pp. 3–52.
Gottschling, M., Keupp, H., Plotner, J., Knop, R., Willems, H., Kirsch, M., 2005. Phylogeny ofcalcareous dinoflagellates as inferred from ITS and ribosomal sequence data. Mol.Phylogenet. Evol. 36, 444–455.
Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysisprogram for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98.
Krichnavaruk, S., Loataweesup, W., Powtongsook, S., Pavasant, P., 2005. Optimal growthconditions and the cultivation of Chaetoceros calcitrans in airlift photobioreactor.Chem. Eng. 105, 91–98.
Kutako, M., Powtongsook, S., 2006. Effect of dilution rate and light intensity on growth ofa diatom Chaetoceros calcitrans under continuous cultivation in flat-panelphotobioreactor. Songklanakarin J. Sci. Technol. 28, 965–975.
Laing, I., 1979. Part 2. Recommended procedure for the rearing of Chaetoceros calcitrans.Fish. Res. Tech. Rep. No. 53. Ministry of Agriculture, Fisheries and Food, Lowestoft,pp. 8–12.
9H.F. Kaspar et al. / Aquaculture 420–421 (2014) 1–9
Laing, I., 1985. Growth response of Chaetoceros calcitrans (Bacillariophyceae) in batch cul-ture to a range of initial silica concentrations. Mar. Biol. 85, 37–41.
Loubiere, K., Olivo, E., Bougaran, G., Pruvost, J., Robert, R., Legrand, J., 2009. A newphotobioreactor for continuous microalgal production in hatcheries based onexternal-loop airlift and swirling flow. Biotechnol. Bioeng. 102, 132–147.
Matyash, V., Liebisch, G., Kurzchalia, T.V., Shevchenko, A., Schwudke,D., 2008. Lipid extractionby methyl-tert-butyl ether for high-throughput lipidomics. J. Lipid Res. 49, 1137–1146.
Medlin, L., Elwood, H.J., Stickel, S., Sogin, M.L., 1988. The characterization of enzymaticallyamplified eukaryotic 16s-like rRNA-coding regions. Gene 71, 491–499.
Michels, M.H.A., van der Goot, A.J., Norsker, N.H.,Wijffels, R.H., 2010. Effects of shear stresson the microalgae Chaetoceros muelleri. Bioprocess Biosyst. Eng. 33, 921–927.
Miller, M.R., Quek, S.Y., Staehler, K., Nalder, T., Packer, M., 2012. Changes in oil content,lipid class and fatty acid composition of the microalga Chaetoceros calcitrans over dif-ferent phases of batch culture. Aquac. Res. 1–14.
Miron, A.S., Gomez, A.C., Camacho, F.G., Grima, E.M., Chisti, Y., 1999. Comparative evalua-tion of compact photobioreactors for large scale monoculture of microalgae.J. Biotechnol. 70, 249–270.
Muller-Feuga, A., Moal, J., Kaas, R., 2003a. The microalgae of aquaculture. In: Stottrup, J.G.,McEvoy, L.A. (Eds.), Live Feeds in Marine Aquaculture. Blackwell Science Ltd., Oxford,pp. 206–252.
Muller-Feuga, A., Robert, R., Cahu, C., Robin, J., Divanach, P., 2003b. Uses of microalgae inaquaculture. In: Stottrup, J.G., McEvoy, L.A. (Eds.), Live Feeds in Marine Aquaculture.Blackwell Science Ltd., Oxford, pp. 253–299.
Myklestad, S., Holm-Hansen, O., Varum, K.M., Volcani, B.E., 1989. Rate of release of extra-cellular amino acids and carbohydrates from the marine diatom Chaetoceros affinis.J. Plankton Res. 11, 763–773.
Pronker, A.E., Nevejan, N.M., Peene, F., Geijsen, P., Sorgeloos, P., 2008. Hatcherybroodstock conditioning of the bluemusselMytilus edulis (Linnaeus 1758). Part I. Im-pact of different micro-algae mixtures on broodstock performance. Aquac. Int. 16,297–307.
Ragg, N.L.C., King, N.,Watts, E., Morrish, J., 2010. Optimising the delivery of the key diatomChaetoceros calcitrans to intensively cultured GreenshellTM mussel larvae, Pernacanaliculus. Aquaculture 306, 270–280.
Raghavan, G., Haridevi, C.K., Gopinathan, C.P., 2012. Growth and proximate compositionof the Chaetoceros calcitrans f. pumilus under different temperature, salinity and car-bon dioxide levels. (Retraction of vol. 39, pg 1053, 2008) Aquac. Res. 43, 639.
Richmond, A., 2004. Principles for attaining maximal microalgal productivity inphotobioreactors: an overview. Hydrobiologia 512, 33–37.
Rico-Villa, B., Coz, J.R.L., Mingant, C., Robert, R., 2006. Influence of phytoplankton dietmix-ture on microalgae consumption, larval development and settlement of the Pacificoyster Crassostrea gigas (Thunberg). Aquaculture 256, 377–388.
Robert, R., Chretiennot-Dinet, M., Kaas, R., Martin-Jezequel, V., Moal, J., Le Coz, J.R., Nicolas,J.L., Bernard, E., Connan, J., Le Dean, L., Le Gourrierec, L., Leroy, B., Quere, C., 2004.Amélioration des productions phytoplanctoniques en écloserie de mollusques:caractérisation des microalgues fourrage. IFREMER, DRV/RA/LPI Argenton 144.
Sarkis, S., Lovatelli, A., 2007. Installation and operation of a modular bivalve hatchery. FAOFisheries Technical Paper No. 492. FAO, Rome (pp. 173 p.).
Takano, H., 1968. On the diatom Chaetoceros calcitrans (Paulsen) emend. and its dwarfform pumilus forma nov. Bull. Tokai Reg. Fish. Res. Lab. 100, 35–43.
Tannock, S.J.C., 2006. Improved mass cultivation of the marine diatom Chaetoceroscalcitrans for shellfish hatcheries. BiotechnologyMassey University, PalmerstonNorth 147.
Tredici, M.R., Biondi, N., Ponis, E., Rodolfi, L., Chini Zittelli, G., 2009. Advances inmicroalgalculture for aquaculture feed and other uses. In: Burnell, G., Allan, G. (Eds.), New Tech-nologies in Aquaculture: Improving Production Efficiency, Quality and EnvironmentalManagement. CRC Press, Boca Raton, pp. 610–676.
White, T.J., Bruns, T., Lee, S., Taylor, J.W., 1990. Amplification and direct sequencing of fun-gal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J.,White, T.J. (Eds.), PCR Protocols: A Guide to Methods and Applications. AcademicPress, Inc., New York, pp. 315–322.
