Outdoor phytoplankton continuous culture in a marine fish–phytoplankton–bivalve integrated system: combined effects of dilution rate and ambient conditions on growth rate, biomass and nutrient cycling

Aquaculture 240 (2004) 211 – 231 www.elsevier.com/locate/aqua-online

Outdoor phytoplankton continuous culture in a marine fish–phytoplankton–bivalve integrated system: combined effects of dilution rate and ambient conditions on growth rate, biomass and nutrient cycling Se´bastien Lefebvrea,*, Ian Proberta, Christel Lefranc¸oisb, Je´roˆme Hussenotc a

Laboratoire de Biologie et Biotechnologies Marines, Universite´ de Caen, esplanade de la paix, 14032 Caen cedex, France b International Marine Center (IMC), Localita Sa Mardini 09072 Torregrande-Oristano, Italy c Centre de recherche sur les e´cosyste`mes marins et aquacoles, CREMA (CNRS-IFREMER), BP 5 17137 L’Houmeau, France Received 10 June 2003; accepted 14 June 2004

Abstract Natural phytoplankton populations were cultured in outdoor continuous cultures using fish-farm effluents as the source of nutrients. The dilution rate was assumed to be the integrating factor of phytoplankton growth and biomass development (flux and stock). In this context, the combined effects of (i) dilution rates of the outdoor culture and (ii) ambient conditions were tested on phytoplankton growth, biomass and cycling of the major nutrient elements (C, N and P). Experiments were carried out in outdoor polyester tanks (0.7 m deep), homogenised by gentle aeration. Si/P ratio was balanced at around 5 in the inflow in order to induce diatom domination while maintaining high N and P assimilation by phytoplankton. Nutrient cycling was assessed through analyses of the different forms of particulate and dissolved nutrients in the inflow and the outflow. Culture dilution rates determined the longevity of the culture and the assimilation efficiency

* Corresponding author. Tel.: +33 2 31 56 71 55. E-mail address: [email protected] (S. Lefebvre). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2004.06.022

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of nutrients. Dissolved phosphorus was the most limiting nutrient. The optimal dilution rate was approximately 0.5 day
1 at 10 8C and 1.5 day
1 at 20 8C with a mean diatom biomass of 9 AM P. Under these conditions, 80% of the dissolved nutrients provided to the tanks were transformed, a production of 8 g C m
2 day
1 and an assimilation rate of 0.3 g P m
2 day
1 were recorded. Assimilation by diatoms was the major pathway of nutrient cycling. During the experiment, a bottom sediment developed progressively and this also played an important role in denitrifying the excess dissolved nitrogen in the fish-farm effluent. However, the results showed that diatom biomass can collapse and we hypothesize that this was the consequence of an increase in cellular sinking rates due to cell aggregation under nutrient or light stress. Modelling approaches are needed in future research in order to determine optimal dilution rates taking into account phytoplankton growth rates, nutrient inputs and ambient conditions (e.g. light and temperature). D 2004 Elsevier B.V. All rights reserved. Keywords: Diatom; Phytoplankton; Dilution rate; Fish effluent; Continuous culture; Nutrients; Integrated system

1. Introduction Integrated mariculture has re-emerged as a sustainable option for the production of seafood with the joint advantages of reducing environmental impact and increasing the commercial value of the system (Chopin et al., 2001). Intensive fish monoculture discharges a high proportion of the nutrients contained in the fish feed to the surrounding environment (e.g. Handy and Poxton, 1983). These nutrients could potentially be used by a primary producer in an integrated system. The main goal of such systems is to associate to the core production (carnivorous fish or shrimps) a pair of primary and secondary producers (microalgae and filter feeders or macroalgae and grazers), the latter being considered as valuable products (Neori et al., 2000). The key process is the transfer of the dissolved fish excretion to the secondary producer through the efficient culture of the primary producer, which provides the trophic link between the two higher level organisms. A number of important issues must be addressed in relation to this process. For example, the quality (form and elemental ratio) and quantity of nutrients excreted by fish can influence both the number and diversity of species developed (Arzul et al., 1996) and the nutrient assimilation efficiency and growth rate of primary producers. The quality (species and biochemical composition) and the quantity of the primary producer must be adapted to the feeding requirements of the secondary producer. In addition, mass production of the primary producer must be adapted to the integrated system in both operational and economic contexts (Shpigel and Neori, 1996). In a previous study, Lefebvre et al. (1996) demonstrated the feasibility of the batch production of pelagic diatoms (Skeletonema costatum, Chaetoceros spp.) from natural population assemblages using dissolved fish excretion as a source of nutrients once the Si/ P ratio was adjusted to N5. The nutritive value of diatoms for bivalves is well documented (Enright et al., 1986; Walne, 1970) and S. costatum, for example, is routinely used to feed oysters or clams in controlled nursery and on-growing systems (Rodhouse and O’Kelly, 1981; Roden and O’Mahony, 1984; Robert and Ge´rard, 1999).

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Continuous culture is a common laboratory technique used in studies of phytoplankton physiology under strictly controlled culture conditions (Droop, 1983). Outdoor continuous phytoplankton cultures have been used for nutrient removal from wastewater (Craggs et al., 1997), and for mass production of phytoplankton rich in polyunsaturated fatty acids for use in mollusc hatcheries and nurseries (Grima et al., 1994), but never in the context of integrated systems. Continuous culture is actually one of the most productive ways of mass producing microalgae and the flow-through systems such as those used in integrated mariculture are fully amenable to this type of culture (Neori et al., 2000). Phytoplankton growth rate is dependent on environmental factors, such as nutrient concentrations, but also temperature and light, which are often correlated to each other on a yearly basis. Temperature and light are the major factors defining the maximal possible growth rate in outdoor continuous cultures (Eppley, 1972), while the nutrient concentration acts as a limiting factor. It indeed defines the actual growth rate obtained within the limits set by light and temperature. Temperature and light cannot be directly controlled in this type of culture, potentially leading to unpredictable culture behavior (De Pauw et al., 1983). On the contrary, nutrient concentrations can be manipulated by varying the dilution rate (and if necessary by manual addition of nutrients in the inflow). In view of an integrated system, the success of an outdoor continuous phytoplankton culture is based on the regulation of the dilution rate in order to achieve the desired compromise between growth rate, nutrient assimilation and biomass accumulation. The aim of this study was to assess the effects of dilution rates under different ambient conditions on the biomass, growth and nutrient cycling of outdoor phytoplankton continuous cultures using dissolved fish excretion as the source of nutrients. As ambient conditions are seasonally dependent, one of the tasks was to assess in three different seasons (winter, spring and summer) the optimal dilution rate in which both high productivity and high nutrient assimilation were achieved. More generally, this study was conducted in the perspective of developing an integrated system for sustainable aquaculture (Folke et al., 1998) in which the phytoplankton can be used as a trophic link between fish excretion and the culture of bivalves.

2. Materials and methods 2.1. Experimental procedures 2.1.1. Materials Three experiments were carried out at different times of the year (spring 1997, winter 1998, summer 1998). Effluents from a land-based sea bass fish-farm were used as the source of nutrients (for more information on the fish-farm, see Lefebvre et al., 2001) and as the inoculum. In each experiment, three phytoplankton cultures were conducted simultaneously in outdoor rectangular concrete tanks (1.1 m3, 0.7 m deep, 0.7 m wide) at three different dilution rates. Experiments are identified depending on the season and the dilution rate in the order of increasing dilution rate (i.e. win-low, win-middle and win-high for the winter period experiment). Replicates of the dilution treatments were not conducted since preliminary results showed little difference between tanks for the measured variables

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(s.e.b3%, author’s unpublished data). The culture medium (i.e. the effluent) was sampled in a pond that gathered all of the effluent from the fishponds. A peristaltic pump was used to provide this effluent to the phytoplankton cultures. Dilution rates were measured and regulated once per day. Air was provided to the phytoplankton cultures at a rate of 4.7 (v/ v) per hour. 2.1.2. Protocol At the beginning of each experiment, the tanks were washed completely (all sediment removed) and filled with effluent with sodium silicate solution added to a molar Si/P ratio of 5. Cultures were grown as batch cultures until significant development of phytoplankton biomass. During this phase, phytoplankton biomass was estimated daily by measurement of in vivo fluorescence. Before the end of the exponential phase, the cultures were transferred to continuous mode with three different dilution rates—low, middle, high. The choice of the middle dilution rate was empirically based on the mean growth rate during the batch phase and the relationship between phytoplankton growth rate and temperature (Eppley, 1972). For the two other tanks, the dilution rate was approximately halved or doubled (except spring-high which was only 30% higher than spring-middle for technical reasons). The dilution rates were kept as constant as possible as long as the temperature remained stable. Dilution rates were increased with temperature during the winter experiment. For all treatments and seasons, sodium silicate solution was added continuously with a peristaltic pump in order to maintain a minimum Si/P ratio of 5 in the inflow. 2.2. Analyses 2.2.1. Abiotic parameters Water temperature (8C) and irradiance (AE m
2 s
1) were recorded hourly with a data logger (Licor, Li-1000) as an average of instantaneous measurements. Temperatures are expressed as a daily average, and irradiance as an integration of the values recorded over 24 h (E m
2 day
1). Salinity, oxygen concentration (WTW portable instruments) and pH (Knick portamess 901) were also measured daily. 2.2.2. Particulate material Accuracy for each analysis was measured with the coefficient of variability (c.v.) as the ratio standard deviation to mean on triplicates and expressed as percentage. Chlorophyll a (Chl a) was analyzed in triplicate from filters or sediments using fluorometry (Turner Designs AU-10) after methanol extraction (Aminot and Chaussepied, 1983; c.v. 4.1%). In vivo fluorescence was determined in duplicate after a 15-min dark adaptation. Particulate organic carbon (POC) and particulate nitrogen (PN) were determined by combusting filters or sediments in a Carlo Erba elemental analyser (Model NA1500; c.v. for C 2.0% and for N 1.7%). Particulate phosphorus (PP) was determined in duplicate according to the method of Koroleff and Grasshoff (1983) by alcaline persulphate oxidation for suspended particulate material (c.v. 3.2%) and by HCl oxidation and heating at 450 8C according to Page et al. (1982) for sediments (c.v. 4.0%). Phytoplankton cell identification and counting (five replicates) were conducted using

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either a Malassez or a Nageotte haemocytometer after addition of formaldehyde (0.5% v/v) and Lugol’s iodine (1 drop per 50 ml of seawater). 2.2.3. Nutrients Dissolved nutrients were sampled after filtration through Whatman GF/F filters (0.7-Am nominal pore size). Total ammonia-N (TAN) and soluble reactive phosphorus (SRP) concentrations were measured in triplicate on fresh samples following Koroleff and Grasshoff (1983; c.v. 2.5%). Water samples were frozen and analyzed within 15 days for silicate (Si, c.v. 3.8%), total dissolved nitrogen (TDN, c.v. 3.5%) and total dissolved phosphorus (TDP, c.v. 4.5%) concentrations using an auto-analyzer (Skalar, Holland), TDN and TDP being analyzed by alcaline persulphate oxydation. Dissolved inorganic carbon (DIC) concentrations were determined in duplicate using total alkalinity according to Strickland and Parsons (1968, c.v. 3.5%). Dissolved organic carbon (DOC) concentrations were determined in duplicate by the UV spectrophotometric method described by Pages and Gadel (1990, c.v. 4.9%). 2.2.4. Meso- and macro-fauna Samples were collected on 50-Am mesh in the inflow and in the outflow water of each tank, and in the sediment on the last day of the experiments. Samples were fixed with 4% (v/v) formaldehyde. Species were determined, counted and the length (L in Am) measured, and mass (m in Ag) estimated according to the Krilov’s equation (Beers, 1985): m=k*L 3. The values for k were 1.510
8 Ag Am
3 for polychaetes, 310
8 Ag Am
3 for calanoid copepods, amphipods and nauplii, and 610
8 Ag Am
3 for harpacticoRd copepods. These values were determined experimentally by weighing a known number of animals (at least 20). The ingestion rate of particulate matter by zooplankton grazers is assumed to have been maximal (I max) due to the high phytoplankton concentration developed in our system (Abu-Rezq et al., 1997). In the literature, I max ranges between 0.35 day
1 (Sciandra, 1986), 1 day
1 (Andersen and Nival, 1989; Raillard and Menesguen, 1994) and 2 day
1 (Carlotti and Radach, 1996) at temperatures of ca 19 8C. We chose the latter value in order to estimate the maximum grazing pressure. When I max is multiplied by the zooplankton biomass (expressed in AM P, 0.4% of dry weight), the flux of grazing on the particulate phytoplankton P biomass is obtained. Dividing the latter by the net production of particulate P gives the grazing pressure in terms of particulate P production. 2.2.5. Water sampling Samples from the inflow and the outflow were taken at the same time each day (sun zenith). At this time of the day, phytoplankton biomass and nutrient concentrations in the inflow and the outflow water were roughly equivalent to the average daily values measured through several 24-h cycles (data not shown). All parameters were recorded daily except Chl a concentrations and zooplankton biomass (once every 48 h). TDP, PN, PP, POC and faunal biomass were not determined for the spring experiment. The bottom sediment of tanks was sampled on the final day of the winter and summer experiment (nutrient budgets could be calculated only for these two experiments).

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2.3. Calculations and statistics 2.3.1. Budget of macro-elements by experiment The value of the budget of a compound X in the inflow (X in) or in the outflow (X out) was expressed as an average flux per day over the experiment (mmol day
1; Eq. (1)). F was the daily water flow rate (m3 day
1). [X] was the concentration of compound X. The first day and the last day of the experiment were noted d 0 and d f respectively; n was the number of days. df P

¯¼

X

½ X i TFi

i¼d0

n

ð1Þ

2.3.2. Phytoplankton daily growth rate An estimation of the phytoplankton daily growth rate (units day
1) over time (t) was calculated from the cell number (X) according to Eppley’s relation for phytoplankton continuous cultures expressed in a logarithmic base. A correcting factor was added to account for phytoplankton cells flowing into the system (X 0) with effluent water (Eq. (2)). D was the daily dilution rate (day
1).     Xt 1 X0t þD 1
l ¼ Ln ð2Þ Xt
1 ðt
t
1 Þ Xt In ambient conditions, light and temperature are often correlated. The data shown are the growth rate maxima for each degree class of temperature. An exponential function was fitted to these observations. These data were also used to feed an empirical model for phytoplankton production (Brush et al., 2002; Eq. (3)) for comparison with our exponential model.       ef E0 E0 ð0:063T Þ
kz exp
0:85 G ¼ 0:974e e
exp
ð3Þ kz Eopt Eopt Daily production was computed by multiplying the maximum daily growth rate by a term to account for photoperiod and light limitation where G is the daily growth rate (day
1), f is the photoperiod (limiting function varying between 0 and 1), k the extinction coefficient (experimental value=4 m
1), z is the depth (m), E 0 is the surface irradiance (PAR, E m
2 day
1), and E opt is the optimal irradiance for photosynthesis (12.5, 15 and 16.5 E m
2 day
1 for the winter, spring and summer experiments, respectively). 2.3.3. Statistics The effect of dilution rates within an experiment was tested using one factor repeated measures ANOVA after logarithmic transformations to account for normality and homogeneity of variance of variables (Von Ende, 2001). This simple repeated measures design was used to determine whether different treatment levels (the dilution rate) applied to the same type of subjects (the tanks) have a significant effect on variables such as fluxes and concentrations over time (see Table 1). When significantly different variances were

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Table 1 General design of the one-way repeated-measures ANOVA used in this study Source of variance

Degree of freedom

Dilution rate (Between-subjects factor)

i
1

Time (Within-subjects factor)

j
1

Sum of squares P SM ¼ ðyi
y¯Þ2 i; j

SB ¼

P

ðyj
y¯Þ2

i; j

Residual Total

(i
1)*( j
1) (i*j)
1

SR = Syy
SB
SM Syy ¼

P

Mean of squares MM ¼ SM i
1

F F ¼ MM MR

MB ¼ SB j
1 MR ¼

SR ði
1Þðj
1Þ

ðyij
y¯Þ2

i; j

This type of ANOVA is equivalent to a randomized-block design in which each individual is considered as a different subject (tanks) on which measurements are repeated over time. Time is the within-subjects factor and the dilution rate is the between-subjects factor. For each subject (tanks), there is only one observation at each modality of the within-subject factor (time). F can be read in the Fisher–Snedecor table to test the hypothesis of equality between the treatment at the risk level a=0.05 (F 0.05 i-1, (i-1)(j-1)).

found, means were ranked using the Student–Newman–Keuls test. Statistics were conducted using Sigmastat 2.0 software. 3. Results 3.1. Time-course measurements Daily average temperature increased steadily from winter to summer, whereas spring and summer integrated irradiances were similar, both being higher than in winter (Table 2). Temperature conditions were more constant than light conditions, the latter sometimes showing high variations (up to 50%) from 1 day to the next in all seasons, and particularly in spring (Fig. 1A). The three experimental dilution rates were significantly different within each season (Table 2). The highest dilution rates applied were in the summer experiment and the lowest in winter (Fig. 1B). Since it was hypothesized that phosphorus was likely to be the most limiting and conservative nutrient (see below), the results presented here focus on this variable. SRP concentrations in the tanks were inversely correlated with the phytoplankton biomass ( Pb0.05, r=
0.55, n=125, Fig. 1C and D). Phosphorus uptake was a function of the biomass (in terms of in vivo Chl a) growing in the system. No significant differences were observed for SRP concentrations in the outflow between the dilution rates ( Pb0.05). The highest biomass typically developed in low dilution rates (Fig. 1D). However, in low dilution rates, the phytoplankton cultures often collapsed dramatically, as for instance in spring-low, spring-middle, sum-low and sum-middle (Fig. 1D). Each of these episodes was preceded by a period (of variable duration) of relatively high extracellular nutrient (mainly P) stress (Fig. 2B–D). For high dilution rates, the biomass was sometimes not high enough to consume SRP, but seemed to self-maintain over longer periods (spring high and sum-high; Fig. 1). It should be noted that SRP concentrations in the inflow fluctuated widely in the spring and summer experiments (Fig. 1C).

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Table 2 Mean (S.E.) characteristics of the winter, spring and summer experiments Dilution rates

Low

Middle

High

Winter Duration Dilution rate Temperature PAR C/Chl a Chl a/cell Si/P l

9 0.25 11.10 33.50 56.50 7.60 4.70 0.390

(0.0)c (0.6)ns (2.4) (2.9)a (0.7)b (0.2) (0.07)c

9 0.48 11.00 id. 38.90 6.80 4.50 0.61

(0.0)b (0.6)ns (2.5)b (0.3)b (0.3) (0.07)b

9 0.90 10.50 id. 35.80 9.20 5.90 1.16

(0.1)a (0.5)ns (1.3)b (0.5)a (0.5) (0.11)a

Spring Duration Dilution rate Temperature PAR C/Chl a Chl a/cell Si/P l

17 0.45 16.50 45.10 n.d. 8.70 5.00 0.20

(0.0)a (0.4) (0.26)c

17 0.68 16.50 id. n.d. 6.70 4.90 0.43

(0.4)b (0.1) (0.12)b

17 0.85 16.70 id. n.d. 6.40 4.90 0.88

(0.0)c (0.4)ns (1.6) (5.0)ns (0.9)ns (1.5) (0.25)c

18 0.70 20.90 46.10 41.40 8.80 8.60 0.78

(0.0)b (0.4)ns (2.3) (6.5)ns (0.7)ns (0.9) (0.16)b

18 1.44 20.90 id. 41.40 8.40 7.30 1.28

(0.0)c (0.5)ns (2.1)

(0.0)b (0.5)ns

(0.0)a (0.5)ns

(0.4)b (0.0) (0.06)a

Summer Duration Dilution rate Temperature PAR C/Chl a Chl a/cell Si/P l

11 0.45 22.10 50.90 37.30 9.70 7.90 0.40

(0.0)a (0.3)ns (8.3)ns (0.7)ns (0.9) (0.18)a

Duration (day), dilution rates (day
1), temperature (8C), P.A.R. (mol E m
2 day
1), C/Chla (Ag Ag
1), Chla/cell (Ag 10
7 cell) and l growth (day
1). One-way repeated measures ANOVA were applied to test for significant differences between the treatments (dilution rate) for each experiment at Pb0.05. Means not sharing a common superscript were significantly different at Pb0.05 (post hoc Student–Newman–Keuls test).

For all experiments, the biomass was not maintained in the system for much longer than 15 days. The maximum biomass was between ca. 10 and 15 relative units of fluorescence corresponding to ca. 230 Ag Chl a l
1 (Chl a=18.72*fluo, Pb0.001, n=80, r 2=0.91). This correlated with an average of 9 AM of particulate P even if SRP concentrations in the inflow were higher. Nevertheless, there were some exceptions such as, for example, on spring-low day 11 or at the beginning of the summer experiment where particulate P Fig. 1. Culture monitoring throughout the winter, spring and summer experiments (A) PAR (vertical bar) and temperature (dark line). (B) Dilution rates, low (white square), middle (grey square), high (black square) (C) SRP in inflow (full black line) and in outflow (dashed line with square). (D) In vivo fluorescence of Chl a in the inflow (full black line) and the outflow (dashed line with square).

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Fig. 2. (A) Monitoring of Si/P ratio in the inflow. Monitoring of nutrient (P,Si) limitation effects calculated using the relationships min[ ([PO4]/([PO4]+kP); ([Si(OH)4]/([Si(OH)4]+kSi)] with kP=0.7 Amol l
1 (Tarutani and Yamamoto, 1994) and kSi=1 Amol l
1 (Andersen and Nival, 1989) black circle P limitation, white circle Si limitation. (B) Low dilution rates. (C) Middle dilution rates. (D) High dilution rates.

reached between 15 and 20 AM (Fig. 1C). TDN concentrations never decreased to concentrations below 10 AM in any of the experiments at any time (data not shown). The addition of silicates was effective in maintaining the molar Si/P ratio in the inflow approaching or greater than 5 except during a period in the spring experiment (Fig. 2A). This can be seen in Fig. 2B where the limiting nutrient was SRP (PO43
) for most of the time, but this limitation fluctuated with silicates at the end of the experiment. 3.2. Phytoplankton growth rates and species composition The maximum daily growth rates (l max) for each temperature class (1 8C) are shown in Fig. 3 where l is plotted against temperature when assimilation efficiency of SRP was greater than 70%. An exponential regression was fitted: l max=0.35*exp(0.077T)

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Fig. 3. Maximum phytoplankton daily growth rate for each degree class of temperature. The black line is the fitting of an exponential model l max=0.35*exp(0.077*T8) (r 2=0.85, n=15, Pb0.001). The grey line is the fitting of Brush et al. (2002) empirical model for estimating phytoplankton production run for the same conditions of light and temperature.

( Pb0.001, r 2=0.85, n=15). This regression showed that a Q 10 of ca. 2 was observed between 10 and 20 8C. This regression did not differ from the estimation of Brush et al.’s (2002) empirical model for phytoplankton production. Phytoplankton growth rate was significantly different between the treatments (dilution rates) at each season: the higher the dilution rate, the higher the phytoplankton growth rate (Table 2). Phytoplankton populations were dominated by centric diatoms and particularly S. costatum in all experiments (N90% numerical dominance). Thalassiosira spp. and Chaetoceros spp. were also observed. 3.3. Meso- and macro-fauna biomass and species In the water column, 70% of total biomass was often comprised of harpacticoid copepods (Euterpina sp., Tisbe sp., Amonardia sp., Harpacticus sp.) in both the inflow and the outflow. Polychaete larvae, copepod nauplii and calanoid copepods (Calanus sp.) constituted the remaining biomass. There were no consistent correlations between the evolution of zooplankton biomass and that of phytoplankton or dilution rates (Fig. 4A and B). The highest zooplankton biomass concentrations in the outflow were similar in winter and in summer although high concentrations were attained more rapidly in winter. This was not the case in the inflow in which zooplankton concentrations were two to three times higher in winter than in summer. In the sediment, harpacticoid copepod biomass was relatively low from two points of view. Firstly, the proportions found in the water column were much higher (N80%) than in the sediment. Secondly, the major biomass was comprised of benthic species, both

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Fig. 4. Zooplankton biomass during the winter and summer experiments in the inflow (full lines) and in the outflow (dashed lines), low dilution rate (white square), middle (grey square) high (black square).

polychaetes (Nereis sp.) and amphipods (Corophium sp.). The sum of the latter two increased concomitantly to dilution rates for each experiment (Table 3). The grazing pressure was calculated on days immediately preceding episodes of biomass collapse. On day 5 of sum-low, the grazing pressure was 7%, on day 8 of summiddle 18%, and on day 14 of sum-high 10%. 3.4. Nutrient fluxes and budget All fluxes in the inflow and in the outflow showed significant differences between the treatments (dilution rates) except for SRP outflow in summer (Tables 4, 5 and 6 ), highlighting the importance of the culture dilution rate in controlling nutrient cycling. 3.4.1. Phosphorus Phosphorus was assumed to be a conservative element throughout each experiment. This was verified in the winter and summer experiments in which total recovery of phosphorus, i.e. the sum of the phosphorus in the sediment and in the outflow compared to the sum in the inflow, ranged from 94% to 105% (Table 4). In the inflow, most of the phosphorus forms were dissolved with TDP representing ca. 80% of total phosphorus inflow, the rest being phosphorus contained in fish faeces (Table 4). Mean concentrations of SRP were approximately 8 AM, with slight differences between experiments even if the amplitude of the variations varied with seasons (Fig. 1, see

Table 3 Whole mass (mg) of benthic meso- and macro-fauna at the end of the winter and summer experiments Copepods (A) Amphipods (B) Polychaetes (C) B+C

Win-low

Win-middle

Win-high

Sum-low

Sum-middle

Sum-high

42.6 75.6 226.5 302.1

109.5 53.1 351.1 404.2

59.3 65.7 375.6 441.3

19.8 152.5 546.3 698.8

111.2 943.2 756.3 1699.5

275.8 1795.4 566.8 2362.2

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Table 4 Phosphorus budget Experiment

Win-low

Duration Dilution rate

9 0.25

9 9 11 18 (0.02)c 0.48 (0.03)b 0.90 (0.07)a 0.45 (0.02)c 0.70

(0.02)b

18 1.44 (0.05)a

Inflow TDP Inflow SRP Inflow PP

1.9 1.8 0.6

(0.1)c (0.1)c (0.0)c

3.5 3.4 1.2

(0.2)b (0.2)b (0.1)b

6.7 6.4 2.2

(0.5)a (0.4)a (0.1)a

6.0 4.6 1.2

(0.4)c (0.3)c (0.1)c

8.4 7.3 1.7

(0.5)b (0.3)b (0.1)b

17.7 15.3 3.6

(1.0)a (0.7)a (0.1)a

Outflow TDP 0.2 Outflow SRP 0.1 Outflow PP 1.9

(0.0)c (0.0)c (0.1)c

0.4 0.1 3.9

(0.1)b (0.0)b (0.2)b

2.4 1.2 5.6

(0.3)a (0.2)a (0.4)a

0.9 0.2 6.1

(0.1)b (0.0)ns (0.6)c

2.3 1.3 6.5

(0.3)b (0.2)ns (0.4)b

4.0 2.5 15.0

(0.3)a (0.3)ns (0.7)a

Initial stock P Final stock P Sediment Recovery

8.9 9.3 5.0 105%

Win-middle

11.0 9.1 6.5 101%

Win-high

9.6 10.0 6.6 98%

Summer-low

18.3 4.4 10.0 94%

Summer-middle

18.7 9.7 24.9 96%

Summer-high

17.6 13.3 27.0 95%

Mean (S.E.) of phosphorus flows (mmol day-1) and budget for winter and summer experiments. Duration of the experiments (day) and dilution rates (day-1). One-way repeated measures ANOVA was applied to test for significant differences between the treatments (dilution rate) for each experiment at Pb0.05. Means not sharing a common superscript were significantly different at Pb0.05 (post–hoc Student–Newman–Keuls test). TDP: total dissolved phosphorus, SRP: Soluble reactive phosphorus, PP: particulate phosphorus.

culture monitoring above). The proportion of DOP ranged from 2% in winter to 30% in summer. In the outflow, the ratio of dissolved and particulate forms was inverted since most of the dissolved forms disappeared with a removal efficiency generally greater than 70% (Table 4). This removal efficiency typically increased when dilution rates of the system decreased. The phosphorus in sediment varied widely from 8% to 29% of total phosphorus inflow (Table 4). 3.4.2. Nitrogen In the inflow, PN represented ca. 10% of total nitrogen. TAN represented 60% to 75% of TDN (Table 5). Average TDN concentrations ranged from 164 to 226 AM (data not shown). In the outflow, TDN removal varied within the same range and in the same way as TDP removal (Tables 4 and 5). However, a significant part of the total nitrogen inflow was lost by the system in each experiment (19% to 29%; Table 5). TAN was the first nitrogen component consumed by the system with TAN in the outflow approaching zero most of the time while other sources of nitrogen (TDN) were still present at significant concentrations (Table 5). 3.4.3. Carbon The carbon inflow was characterised by the low ratio of dissolved carbon (DIC and DOC) to POC (Table 6) compared to nitrogen and phosphorus budgets (Tables 4, 5 and 6). However, DIC was three times higher in summer (285 AM) than in winter (95 AM; data not shown). In the outflow, DIC was much lower than in the inflow and there was a slight increase in DOC. The concentration of POC was inversely proportional to the dilution rates, ranging from 400 to 818 AM (data not shown). Two to four times more carbon was

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Table 5 Nitrogen budget Experiment Duration Dilution rate

Win-low 9 0.25 (0.02)c

Win-middle 9 0.48 (0.03)b

Win-high

Summer-low

Summer-middle Summer-high

9 11 18 0.90 (0.07)a 0.45 (0.02)c 0.70 (0.02)b

18 1.44 (0.05)a

Inflow TDN Inflow TAN Inflow PN

36.8 (2.1)c 25.2 (1.5)c 6.3 (0.4)c

73.4 (5.5)b 138.4 (10.9)a 100.7 (6.7)c 148.4 50.0 (3.7)b 94.7 (7.7)a 87.8 (7.5)c 134.4 12.2 (0.8)b 23.2 (1.7)a 14.8 (0.8)c 21.1

(8.3)b (8.5)b (0.8)b

313.8 (17.5)a 284.3 (17.3)a 44.1 (1.6)a

Outflow TDN Outflow TAN Outflow PN

5.4 (0.4)c 0.0 (0.0)b 23.2 (1.7)b

8.4 (0.5)b 0.1 (0.0)b 51.6 (3.2)a

(3.6)b (2.6)b (6.2)b

117.9 (10.1)a 65.0 (7.6)a 174.7 (7.7)a

Initial stock N Final stock N Sediment Recovery

118.1 101.8 55.2 71%

117.8 116.3 63.5 74%

47.3 11.1 70.7

(5.5)a (3.3)a (6.5)a

118.6 92.6 40.4 73%

21.7 (2.6)b 4.9 (0.9)b 79.4 (7.8)c 322.5 60.3 79.3 74%

42.7 17.3 95.3 252.8 127.5 164.0 79%

259.1 128.4 195.0 81%

Mean (S.E.) of nitrogen flows (mmol day-1) and budget for winter and summer experiments. Duration of the experiments (day) and dilution rates (day-1). One-way repeated measures ANOVA was applied to test for significant differences between the treatments (dilution rate) for each experiment at Pb0.05. Means not sharing a common superscript were significantly different at Pb0.05 (post–hoc Student–Newman–Keuls test). TDN: total dissolved nitrogen, TAN: Total ammonia nitrogen, PN: Particulate nitrogen.

present in the outflow compared to the total inflow (recovery 117% to 225%; Table 6), indicating that most of the carbon was fixed from the air.

Table 6 Carbon budget Experiment Duration Dilution rate

Win-low 9 0.25 (0.02)c

9 0.48 (0.03)b

Win-high 9 0.90 (0.07)a

Summer-low 11 0.45 (0.02)c

Summer-middle Summer-high 18 0.70 (0.02)b

18 1.44 (0.05)a

(3.1)b 133.5 (7.3)a (2.3)b 82.3 (4.5)a (4.8)b 140.6 (10.5)a

78.8 (4.6)c 154.9 (7.2)c 104.3 (5.6)c

122.3 (5.1)b 260.1 (10.1)b 145.5 (5.5)b

256.5 (11.3)a 533.6 (23.0)a 302.9 (11.3)a

Outflow DOC 50.8 (3.1)c 85.5 (4.5)b 154.2 (8.8)a Outflow DIC 2.7 (0.2)b 4.4 (0.3)b 10.3 (0.8)a b Outflow POC 196.3 (14.1) 379.6 (23.1)a 438.3 (38.4)a

104.1 (6.2)c 3.2 (0.2)b 485.8 (48.3)c

141.6 (5.7)b 7.1 (0.5)b 581.2 (38.7)b

275.1 (11.3)a 14.7 (0.7)a 955.0 (43.1)a

Inflow DOC Inflow DIC Inflow POC

38.4 22.9 38.3

Initial stock C 881.4 Final stock C 900.0 Sediment 391.3 Recovery 199%

(2.3)c (1.1)c (2.5)c

Win-middle

71.0 44.3 74.0

754.0 904.3 390.2 225%

548.1 499.5 288.0 165%

1085.9 450.3 554.5 157%

1047.2 687.7 1064.6 141%

1033.8 355.1 1423.4 117%

Mean (S.E.) of carbon flows (mmol day-1) and budget for winter and summer experiments. Duration of the experiments (day) and dilution rates (day-1). One-way repeated measures ANOVA was applied to test for significant differences between the treatments (dilution rate) for each experiment at Pb0.05. Means not sharing a common superscript were significantly different at Pb0.05 (post hoc Student–Newman–Keuls test). DOC: dissolved organic carbon, POC: Particulate organic carbon.

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3.5. Molar ratio and cell composition The atomic N/P (all forms) ratio (mol mol
1) in the inflow was almost constant throughout each experiment and between each experiment with an average value of 17.1 ( Pb0.05; Fig. 5A). The N/P atomic ratio (particulate forms) in the outflow was lower and averaged 12.2 for all experiments ( Pb0.05; Fig. 5A). Total particulate carbon in the outflow was not a linear function of the inflow phosphorus, reaching a plateau at high dilution rates (Fig. 5B). This was particularly clear in winter experiments in which TPC

Fig. 5. (A) Linear relationship between total phosphorus and total nitrogen in the inflow and particulate nitrogen and particulate phosphorus in the outflow ( Pb0.05). (B) Total phosphorus in inflow plotted against particulate carbon in outflow.

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was lower in win-high than in win-middle. The Chl a/cell values ranged from 6.4 to 9.7 Ag Chl a 10
7 cells with no relationship with dilution rates. The highest Chl a/cell values occurred at low dilution rates for the spring experiment and at high dilution rates for the winter experiment, whereas there were no significant differences between treatments for the summer experiment (Table 2). The cell concentrations produced were of the same order for the winter and spring experiments (200–250 cell Al
1), whereas they were higher for the summer experiment (300–400 cell Al
1).

4. Discussion The results of this study demonstrate that outdoor continuous cultures of phytoplankton can be effective in converting dissolved nutrients from fish-farm effluent into particulate matter (mainly diatoms), which would be suitable for bivalve production. Manual adjustment of the molar Si/P ratio (Si/PN5) in the inflow favoured the production of diatoms (in our study particularly S. costatum), an observation previously reported by several authors using outdoor continuous cultures (Dunstan and Tenore, 1974; Harrison and Davis, 1979; Roden and O’Mahony, 1984). Continuous culture of phytoplankton differs conceptually from the culture of macroalgae which has also been used in integrated systems (Neori et al., 2000). In the latter, the biomass is usually more constant over time in the system, whereas phytoplankton biomass is washed out of the system at the same rate as nutrient inflow with the renewal of water. Thus, the system is more time-dynamic in terms of biomass and therefore more sensitive to changes in the parameters controlling phytoplankton growth rate. For this reason, we surveyed a variety of different forms of nutrients that can influence phytoplankton growth rate, whereas most previous studies of integrated systems have focussed on ammonia nitrogen (e.g. Neori et al., 1991 or Porello et al., 2003). 4.1. Fish-farm effluent as culture medium Concentrations and relative proportions of the major elements (N, P) in the inflow were favourable for the production of microalgae. The N/P molar ratio was higher than the assimilated Redfield ratio for microalgae (16). Several authors have indicated the optimal assimilated N/P molar ratio for S. costatum close to 12 in non-limiting nutrient conditions (Harrison et al., 1977; Burkhardt et al., 1999), a value recorded in the culture outflow in our experiments. A N/P ratio of ca. 17 in the fish-farm effluent was similar to values reported for farmed sea bass (Lemarie´ et al., 1998), and sea bream (Lupatsch and Kissil, 1998). Concentrations of N and P in the inflow to the experimental tank were dependent on fish excretion rates and water inflow rate. Importantly, dissolved N and P concentrations were compatible with the equivalent concentrations of particulate phytoplankton N and P that it is possible to produce (i.e. 8 to 10 AM P) without affecting phytoplankton growth rate by production of auto-inhibitive substances (Imada et al., 1992). Silicate was shown to be typically the most limiting of macro-nutrients in fish-farm effluents (Lefebvre et al., 1996). When the Si/P ratio was maintained at ca. 5 in the inflow in our experiments, nutrient limitation fluctuated between dissolved P and Si with a predominance for P limitation (Fig. 2B–D) which can therefore be considered as the main

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limiting nutrient of the system. Analyses of particulate phytoplankton confirmed that P was the most limiting nutrient since both C/P (70–100) and N/P (12) ratios were higher than described in the literature for the observed diatom species in non-limiting nutrient conditions (Harrison et al., 1977; Burkhardt and Riebesell, 1997). DIC concentration in the system, which remained above 5 AM, did not seem to limit diatom growth rate, and microalgal elemental composition did not reflect any DIC stress (Burkhardt et al., 1999). Non-limiting DIC concentrations should be favoured by CO2 excretion by fish and by aeration. 4.2. Nutrient assimilation and growth rate In our continuous cultures, nutrient assimilation was directly correlated with phytoplankton growth rates and biomass, as is characteristic of this type of culture (see review by Droop, 1983) . The difficulty in using outdoor cultures is to maintain a dquasi steady stateT in which a compromise is reached between biomass level and nutrient concentration (nutrient assimilation should be as high as possible without reducing nutrients to such a point that growth rate is too strongly affected). Biomass development was a balance between algal growth and dilution rates of the system. High dilution rates (=high nutrient concentration) allowed high growth rate but sometimes lower biomass and therefore lower nutrient assimilation. In contrast, low dilution rates allowed higher biomass (and nutrient assimilation), but resulted in lower growth rate. For given nutrient concentrations, the optimal way in which to run the system could be based on empirical relationships of the maximum growth rate observed for each class of temperature in the experiments. The relatively good fitting of Brush et al.’s (2002) empirical model of primary production for phytoplankton (Fig. 3) indicates that there was no growth inhibition by dexternalT factors, i.e. factors other than temperature (and hence light due to the yearly correlation of these two factors) and nutrient concentration when using fish-farm effluent as the culture medium. The latter observation cannot be generalised, however, since the level of intensification of fish-farming (the use of therapeutic substances for example) and the phytoplankton population composition (Arzul et al., 1996) must be taken into account. In our experimental conditions, a maximum growth rate of 1.5 day
1 was attained at 20 8C, which, with a mean of 9 AM P of phytoplankton biomass and 0.7-m water depth, allowed a removal efficiency of 0.3 g P m
2 day
1 and a production of 8 g C m
2 day
1 (with a C/P molar ratio of 70). As the empirical relationship showed a Q 10 of 2, these results are halved at 10 8C. Such an assimilation rate was possible because of the high growth rate and high P requirements of S. costatum (Finenko and Krupatkina-Akinina, 1974). Under high dilution rate conditions, 80% of P could be assimilated by phytoplankton, reducing dissolved P concentration to around 1 AM. Cellular chlorophyll a content (Chl a or C/Chl a ) indicated that light limitation very likely occurred in our outdoor system since Chl a content was higher than observed in previous studies (Harrison et al., 1977, Sakshaug and Andresen, 1986). Light is considered the most limiting factor in outdoor systems (Oswald, 1988), and this seems to have been the case even with the high assimilation efficiency and therefore low nutrient concentrations observed in our system.

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4.3. Biomass collapse In our experiments, dramatic collapses in the phytoplankton population periodically occurred. Two phenomena may be relevant in this context: grazing by meso- and macrofauna and sedimentation processes under nutrient stress. Our estimations of grazing pressure, which were based on the maximum ingestion rate reported in the literature (2 day
1, Carlotti and Radach, 1996), indicate that although meso- and macro-fauna exerted a significant grazing pressure (up to 18%), this alone was not likely to have been responsible for the biomass collapse during the summer experiment. Zooplankton biomass concentrations increased with time, particularly at low dilution rates. This might suggest that zooplankton production was higher than dilution rates. The maximal zooplankton growth rate calculated by a modelling approach was 0.4 day
1 at 20 8C (Raillard and Menesguen, 1994). This growth rate does not exceed the minimum dilution rate used in our experiments. Zooplankton may differentially remain in the system using active vertical migration towards the sediment at night. Although this process was not measured, we suspect that it contributed significantly to increases in zooplankton biomass over time. Whether as a result of growth or active migration, increases in zooplankton biomass (and hence grazing pressure) may become a relevant factor over medium to long time-scales (N20 days), longer than the duration of our experiments. As for sedimentation processes, an increase in the settling velocity has been reported to occur with ageing of the culture (Andersen and Nival, 1989). Jorgensen (1989) reported a settling velocity up to 1.35 m day
1 for S. costatum. Thornton and Thake (1998) showed that an increase in settling velocity occurred due to cell aggregation with increasing temperature and under nutrient limitation. The fact that nutrient concentrations can vary in the inflow could favour sporadic nutrient stress (as seems to be indicated in Fig. 2B–D) and sedimentation. This highlights the importance of regulating inflow rate according to fishpond nutrient concentrations in order to maintain nutrient concentrations as stable as possible. 4.4. Nitrogen loss through gas Our calculations indicate a large nitrogen loss from the system which can be attributed to exchange with the atmosphere. Most authors using microalgal production in wastewater tertiary treatment have attributed this loss to ammonia volatisation (e.g. Oswald, 1988). Porello et al. (2003) came to the same conclusion in the context of phyto-treatment ponds (macroalgae) used to remove ammonia from land-based aquaculture waste. Ammonia nitrogen was often at low concentrations in our experimental tanks, while nitrate concentrations were relatively high. We suggest, however, that this was due to the assimilation of ammonia by microalgae rather than volatilisation. Ammonia nitrogen is often the first nitrogen compound consumed by microalgae because of its reduced state (Levasseur et al., 1990). At the pH measured in our experimental system (maximum 8.5), dissociation equations dictate that most ammonia nitrogen was in the form of ammonium ions (NH4+), which are easily assimilable by algae, rather than ammonia (NH3) which is volatile. We suggest that denitrification was the key process of nitrogen loss in our system

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because of the coupling of high organic matter levels in the sediment (=high bacterial activity and thus high oxygen consumption), oxidised conditions at the sediment–water interface (necessary for nitrification) and anaerobic conditions of sub-surface sediment layers which leads to denitrification (De Vries and Hopstaken, 1984). The fact that nitrogen loss, as well as organic matter concentrations in the sediment were proportionally higher in low dilution rates (data not shown) supports this statement. Furthermore, the estimation of nitrogen loss as gas in this study is within the same range as that reported in hyper-nutrified estuaries (Olgivie et al., 1997). 4.5. Future perspectives Outdoor continuous cultures of phytoplankton provide a system in which the dissolved excretion from fishponds may be transformed from dissolved inorganic material into particulate phytoplankton organic material. However, a method for maintaining biomass over longer periods in the system is needed. De Pauw et al. (1983) demonstrated the possibility of maintaining outdoor phytoplankton continuous cultures in a healthy state for several months by the manual manipulation of nutrient levels in the inflow. Continuous adaptation of inflow rate to conditions of light, temperature and nutrient concentrations is certainly necessary. This control could (i) prevent grazers from accumulating in the system (Rusch and Malone, 1998), (ii) avoid sporadic periods of phytoplankton nutrient stress and sinking, and (iii) improve nutrient assimilation by phytoplankton on longer time scales. A modelling approach is required to predict and control the dilution rates of the cultures taking into account the nutrient budget of the whole integrated system and particularly that of the fish compartment providing the nutrients for the phytoplankton culture (Lefebvre et al., 2001). The practicalities of such an approach are being assessed in an ongoing European Innovative Project, dGenesisT (Hussenot and Shpigel, 2003), with the objective of developing reliable protocols for the management of viable outdoor fish–phytoplankton–bivalve integrated culture systems.

Acknowledgements We would like to thank Marcel Guillaut, Lucette Joassard, Franc¸oise Mornet and Michel Prineau for the technical assistance. We would also like to thank Dr. Jean-Luc Mouget, for interesting comments. Special gratitude is extended to the private farm company and its managers Bernard Houin and Andre´ Zwaga, who allowed us access to their facilities.

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