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Carbon cycling in a zero-discharge mariculture system

Kenneth Schneider a,b,*, Yonatan Sher a,1, Jonathan Erez b, Jaap van Rijn a

aThe Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, Department of Animal Sciences,

P.O. Box 12, Rehovot 76100, IsraelbThe Hebrew University of Jerusalem, The Institute of Earth Sciences, Edmond Safra Campus, Givat Ram, Jerusalem 91904, Israel

a r t i c l e i n f o

Article history:

Received 16 September 2010

Received in revised form

8 December 2010

Accepted 25 January 2011

Available online 21 February 2011

Keywords:

Zero-discharge mariculture

Carbon stable isotopes

Alkalinity

DIC

* Corresponding author. Present address:CA 94305, USA.

1 Current address: The Jacob Blaustein Ins84990, Israel.0043-1354/$ e see front matter ª 2011 Elsevdoi:10.1016/j.watres.2011.01.021

a b s t r a c t

Interest in mariculture systems will rise in the near future due to the decreased ability of

the ocean to supply the increasing demand for seafood. We present a trace study using

stable carbon and nitrogen isotopes and chemical profiles of a zero-discharge mariculture

system stocked with the gilthead seabream (Sparus aurata). Water quality maintenance in

the system is based on two biofiltration steps. Firstly, an aerobic treatment step comprising

a trickling filter in which ammonia is oxidized to nitrate. Secondly, an anaerobic step

comprised of a digestion basin and a fluidized bed reactor where excess organic matter and

nitrate are removed. Dissolved inorganic carbon and alkalinity values were higher in the

anaerobic loop than in the aerobic loop, in agreement with the main biological processes

taking place in the two treatment steps. The d13C of the dissolved inorganic carbon (d13CDIC)

was depleted in 13C in the anaerobic loop as compared to the aerobic loop by 2.5e3&. This

is in agreement with the higher dissolved inorganic carbon concentrations in the anaerobic

loop and the low water retention time and the chemolithotrophic activity of the aerobic

loop. The d13C and d15N of organic matter in the mariculture system indicated that fish fed

solely on feed pellets. Compared to feed pellets and particulate organic matter, the sludge

in the digestion basin was enriched in 15N while d13C was not significantly different. This

latter finding points to an intensive microbial degradation of the organic matter taking

place in the anaerobic treatment step of the system.

ª 2011 Elsevier Ltd. All rights reserved.

1. Introduction coastal waters with organic matter and nutrients (Wu, 1995).

The oceans supply of fish is stagnant and it is expected to

decrease in the near future due to overfishing and biodiversity

loss (Jackson et al., 2001; Worm et al., 2006). As such, it is

expected that mariculture systems will have a significant role

in the global fish supply because of the growing demand for

fish (Tidwell and Allan, 2001). Mariculture systems in coastal

areas (such as floating cages) or coastal, land-based ponds

impose environmental concerns due to their contamination of

Department of Global

titute for Desert research

ier Ltd. All rights reserved

Such contamination brings about environmental alterations

such as anoxic sediments that produce toxic H2S (Holmer and

Kristensen, 1992), eutrophication, and a resulting decrease in

biodiversity and biomass of the benthic communities

(Mazzola et al., 1999; Karakassis et al., 2000).

In the present study, a zero-discharge recirculation

system, first developed for freshwater fish farming (van Rijn,

1996; Shnel et al., 2002) and later converted to a system for

culture of marine fish (Gelfand et al., 2003) was examined.

Ecology, Carnegie Institution, 260 Panama street, Stanford,

, The Ben Gurion University of the Negev, Sede Boqer Campus,

.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 2 3 7 5e2 3 8 22376

Water quality in the system ismaintained by recirculating the

culture water through two main treatment loops: an aerobic

loop consisting of a trickling filter (TF) used for oxidation of

ammonia to nitrate by nitrifying bacteria (Chen et al., 2006;

Eding et al., 2006) and an anaerobic loop, consisting of

a digestion basin (DB) and a fluidized bed reactor (FBR), in

which, among other processes, organic matter is digested and

nitrate respired to elemental nitrogen (van Rijn et al., 2006).

The high organic load in the DB creates a redox gradient that

facilitates conditions not only for nitrate reduction but also for

sulfate reduction to harmful sulfide. It was demonstrated that

while in the DB some of the produced sulfidewas reoxidized to

sulfate by autotrophic denitrifiers (Sher et al., 2008), sulfide

oxidation by these organisms was particularly evident in the

FBR, which served as a final sulfide polishing stage before

water from the anaerobic treatment stepwas returned back to

the fish basin (Cytryn et al., 2005).

Stable isotopes can be used to trace natural processes on

the basis of their distribution as different biological processes

result in different isotopic fractionation. Processes such as

photosynthesis and chemosynthesis are associated with

a large discrimination against 13C resulting in low d13C values

of �11 to �35& depending on the process type and the

enzymes associated with them (Guy et al., 1993; Goericke and

Fry, 1994; Robinson et al., 2003). Contrary to other processes,

carbon stable isotopes have little fractionation in the biolog-

ical food web. It was observed that with each trophic level

a slight increase in d13C of about 0.8 � 1.1& takes place;

a phenomenon known as “you are what you eat�1&” (DeNiro

and Epstein, 1978). As opposed to carbon, nitrogen isotopes

have a larger fractionation with each trophic level and

increases in d15N by about 3 � 2.6& have been reported

(DeNiro and Epstein, 1981). The distinctive fractionation of

carbon and nitrogen stable isotopes has been used as tracers

in food web studies in natural systems and in engineered

systems using mixing models based on mass balance

consideration (Schroeder, 1983; Fry and Sherr, 1984).

In this study we present a description of the carbon cycling

in a zero-discharge mariculture system based on carbon and

nitrogen stable isotopes and chemical parameter in the water

and sludge phases of a zero-discharge mariculture system

through measurement of changes in values of d13C and d15N

and changes in concentrations of NH3, NO3�, H2S, alkalinity,

dissolved inorganic carbon (DIC), redox potential and dis-

solved oxygen (DO).

2. Materials and methods

2.1. General description of the mariculture system

The intensive fish mariculture system was an enlarged

version of the system previously described by Gelfand et al.

(2003). The system (Fig. 1) comprised a fish basin (5 m3)

stocked with the gilthead seabream (Sparus aurata) from

which water was recirculated through aerobic and anaerobic

treatment compartments. The aerobic compartment con-

sisted of a trickling filter (TF) with a volume of 8 m3 and

a surface area of 1920 m2. Surface water from the fish basin

(FB) was recirculated through the aerobic compartment at

a rate of 10 m3 h�1. In addition to the TF, the aerobic

compartment comprised a foam fractionator (FF), which

received water from the trickling filter basin (TFB). The

particulate organic matter captured by the FF was discharged

into the digestion basin (DB). This latter basin (volume: 5.4 m3)

was part of the anaerobic treatment compartment. By gravi-

tation, water from the bottom of the FB was led (0.8 m3 h�1)

into this latter basin. Effluent water from the DB was collected

in an intermediate collection basin (ICB) before being

returned, by gravitation, to the TFB. Water from the ICB was

recirculated (0.8 m3 h�1) through a fluidized bed reactor (FBR,

volume:13 L) which effluent water was led through a swirl

separator (SS) before being returned to the ICB. Sludge

captured by the SS, was discharged into the DB. Previous

studies on this and similar systems revealed that nitrification

(aerobic treatment loop), digestion of organic matter together

with nitrate and sulfate respiration (digestion basin), and

microbial sulfide oxidation (FBR), were major processes

affecting the overall water quality in the system (van Rijn

et al., 1995).

2.2. Sampling regime

2.2.1. Sampling frequency and locationsThe mariculture system was sampled on three separate

occasions between July and December 2006. Samples were

withdrawn from nine different locations within the system

(Fig. 1): (1) fish basin (FB), (2) trickling filter collection basin

(TFB), (3) effluent water from the trickling filter (TFout), (4)

influent water to the trickling filter (TFin), (5) top layer (5 cm

depth) of influent zone in the digestion basin (DBinT), (6)

bottom layer (30 cm depth) of influent zone in the DB (DBinB),

(7) top layer (5 cm depth) of effluent zone in the DB (DBoutT), (8)

bottom layer (30 cm depth) of effluent zone in the DB (DBoutB),

and (9) fluidized bed reactor (FBR).

2.2.2. Sampling procedureWater from each sampling point was collected in a 1.5 L

plastic bottle and was initially filtered through cotton gauze

with a mesh size of several mm for removal of agglomerated,

floating sludge particles. Water for chemical and isotopic

analysis was further filteredwith a GF/F or GF/C before storage

as described below. Water for DB chemical profiling of the DB

was collected in 50 ml vials as described below (treatment of

sludge samples). The vials were centrifuged immediately and

the water and sludge where separated for different analysis.

Water for measuring sulfide was transfer to 10 ml vial under

nitrogen environment and the sulfide was fixed immediately

with zinc acetate (Strocchi et al., 1992).

2.2.3. Treatment of water samplesImmediate after collection the pH, redox and inorganic

nitrogen species (NO3�, NO2

� and NH3) and sulfide (S2�) were

analyzed as described below. Samples for alkalinity, dissolved

inorganic carbon (DIC) and carbon stable isotopes of DIC

(d13CDIC) were stored in 60 mL brown glass bottles with gas-

tight screws and refrigerated (4 �C) until measurements. DIC

and d13CDIC samples were poisoned with 0.6 mL (1% v:v) of

saturated HgCl2 solution immediate after sampling. Particu-

late organic matter (POM) from a known water volume was

DB RBF

S

FF

FB

TFB

TF

- Pump

Aerobic loop Anaerobic loop

TFout

TFin

BCI

DBin

DBoutInlet

Outlet

Center

Digestion Basin(DB)

DBinT

DBinB

DBoutT

DBoutB

Sludge

A

B

Fig. 1 e Schematic design of the zero-discharge fish mariculture system (A). Detailed diagram of the digesting basin (B).

Abbreviations are as follows: FB [ fish basin, TF [ trickling filter, TFB [ trickling filter basin, TFin [ influent water to the

trickling filter, TFout [ effluent water from the trickling filter, FF [ foam fractionator, DB [ digestion basin, DBinT [ top

layer (5 cm depth) of influent zone in the digestion basin, DBinB [ bottom layer (30 cm depth) of influent zone in the DB,

DBoutT [ top layer (5 cm depth) of effluent zone in the DB, DBoutB [ bottom layer (30 cm depth) of effluent zone in the DB,

FBR [ fluidized bed reactor, S [ swirl separator and ICB [ intermediate collection basin.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 2 3 7 5e2 3 8 2 2377

collected on combusted (450 �C for 2 h) GF/F filters and dried at

30 �C for about 48 h.

2.2.4. Treatment of sludge samplesSludge from the DB was sampled from three places (inlet,

center and outlet) at 2e4 depths (12 cm apart) depending on

the sludge depth. The samples were collected from different

depths using a sampling device consisting of a 50 ml vial

attached to a scaled pole. Samples were collected by lowering

the closed vial to the desired depth and lifting the lid of the

vials for a few seconds to allow filling of the vial. When

possible, sludge was collected from the wall of the TFB. About

2e3 g of wet sludge was dried at 30 �C for about 48 h.

2.3. Chemical analysis

NH3 and NH4þ, referred to as total ammonia nitrogen (TAN),

were determined by oxidation with salicylate-hypochlorite

method (Bower and Holm-Hansen, 1980). Nitrite was deter-

mined by reactionwith sulfanilamide (Strickland and Parsons,

1972). Nitrate was measured according to the light absorption

at two wave lengths 220 and 275 nm (APHA, 1998). When

sulfide was present in the samples, samples were diluted with

an HCl (0.1 N) solution in 1:1 proportion and nitrate was

measured at an additional wave length of 250 nm accounting

for the HS� remaining in the solution (Sher et al., 2008). Sulfide

was determined by the methylene blue method (Cline, 1969).

Total alkalinity was determined by titration with hydrochloric

acid and calculated according to the Gran titration method

(Grasshoff et al., 1983). Measurements of pH were conducted

with a Radiometer Copenhagen pHmeter (PHM92 Research pH

Meter) and a Radiometer Copenhagen combination electrode

(GK2401c). The electrode was calibrated using NBS scale

standard buffers (Radiometer analytical) of 7.000 and 10.012.

DO and temperature measurements at different sites at the

mariculture system were conducted by means of an oxygen

A

B

C

Fig. 2 e DIC (A), d13CDIC (B) and alkalinity (C) measured at

different locations of the mariculture system,

abbreviations as in Fig. 1. The data is presented as

mean ± SD.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 2 3 7 5e2 3 8 22378

electrode combined with a temperature probe (OxyGuard,

H01c Handy Gamma). Salinity was monitored with a refrac-

tometer (model: S-10E, Atago, Tokyo, Japan).

2.3.1. Stable isotopesStable isotopes are measured relative to an international

standard (eq. (1)) in per mil (&).

d13C or d15Nð&Þ ¼�Rsample

Rstandard� 1

�� 1000 (1)

Where d13C and d15N are the values measured for C and N,

respectively and R is the ratio of the heavier isotope to the light

isotope (13C/12C and 15N/14N). For each element, we used

a commonly used international standard. For C, this standard

is PDB (Pee Dee Belemnite-marine limestone) and for N,

atmospheric air is used as the international standard.

2.3.2. d13CDIC and DIC measurements1-mL samples were injected into a 10-mL vial with a gas-tight

screw filled with He gas at atmospheric pressure. Ten drops of

H2PO4 (85%) were added and the vials were left for 24 h to

equilibrate at 25 �C in a temperature controlled sample tray

(Finnigan�gasbench, Thermo Electro cooperation, USA). In

each sample d13CDIC was measured eight times using an auto-

sampler gas bench system connected online to an isotope

ration mass spectrometer (IRMS; 252 mat, Finnegan) and the

d13CDIC was averaged. The DIC was estimated from the first

measured signal peak (mV) of each sample according to

a calibration curve calculated from samples with a known DIC

concentration, freshly prepared at each day of analysis.

2.3.3. POM, fish tissue, feed pellets and sludge d13C and d15NmeasurementsDried sludge, freeze dried fish tissue and feed pellets were

grained and samples weighing between 150 and 1100 mg,

dependingon their organicmatter content,wereanalyzed.The

filter upon POM sample were collected (as described above)

was divided into quarters. The grounded material or a 1/4 of

filterwerewrapped in tin cups andmeasuredusing an element

analyzer connected online to an IRMS (252 mat, Finnegan).

2.4. Calculations

Carbon concentration in the POM (POMC) was calculated

according to the following equation,

POMC ¼ WCs� 4Vs

(2)

Where POMC is expressed inmg L�1, WCs is the carbonweight

fraction of the organic matter on the filter measured by the

mass spectrometer in mg and multiplied by 4 to account for

using only 1/4 of the filtered material in the analysis, Vs is the

water volume filtered. Nitrogen concentration in the POM

(POMN) was calculated in the same manner as POMC by

replacing WCs with WNs the weight of N in the filter as

measured by the mass spectrometer in mg.

2.4.1. Statistical analysisData are expressed as mean � SD. A comparison between

treatments was performed using the ANOVA test. Statistical

significance was set at a level of 5% or less. Statistica 6 soft-

ware (StatSoft) was used for statistical calculations.

3. Results

3.1. In situ measurements within the mariculturesystem

3.1.1. The inorganic carbon systemThe DIC, alkalinity and the d13CDIC showed distinctive differ-

ences between the aerobic (TF-FB) and anaerobic (DB-FBR-FB)

loops (Fig. 2). DIC in the aerobic loop was in the range of

2900e3900 mmol L�1 with an average of 3400 � 385 mmol L�1,

and was lower than in the anaerobic loop which was in the

range of 4200e5200 mmol L�1 with an average of

A

B

Fig. 3 e (A) A typical depth profile of DO (,) and Redox (C)

in the DB center. (B) A typical depth profile of NOL3 (B),

S2L (:) and TAN (,) measured in the DB center.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 2 3 7 5e2 3 8 2 2379

4630 � 296 mmol L�1. In the FBR, DIC was higher by about

1000 mmol L�1 (Fig. 2a). The high variation in the DIC is mainly

due to a variation between sampling dates. Within each

sampling session, the variation averaged 138 � 51 and

184 � 99 mmol L�1 for the aerobic and the anaerobic loops,

respectively. The average DIC difference between the aerobic

and anaerobic loops was statistically significant (ANOVA,

p < 0.002).

The d13CDIC (Fig. 2b, Table 1) showed 13C enriched values in

the aerobic loop of �8.03 � 0.25& compared to values of

�10.8 � 0.37& in the anaerobic loop. The average difference

between the two loops was statistically significant (ANOVA,

p < 0.005). Alkalinity in the aerobic loop ranged from 4080 to

4260 mmol L�1 with an average value of 4160 � 80 mmol L�1,

significantly lower than the anaerobic loop, which fluctuated

between 4900 and 5600 mmol L�1 with an average of

5300 � 350 mmol L�1 (Fig. 2c).

3.1.2. POMd13CPOM in all the 9 sampling sites of the system was very

similar (Table 1) with an average of �23.05 � 1.08&,

�23.29 � 1.43& and �23.16 � 1.28& for the aerobic, anaerobic

and the all the stations combined, respectively. Carbon

concentrations in POM (POMC) averaged 2.5 � 0.5 and

4.1 � 0.4 mg C L�1 in the aerobic loop and anaerobic loop,

respectively. This difference was statistically significant

(ANOVA, p < 0.03). d15NPOM in the TF was enriched in 15N with

values of around 9& compared to the other sampling sites in

the mariculture system where d15NPOM values were around

7.5& (Table 1). Nitrogen concentrations in the POM (POMN)

were lower in the aerobic loop than in the anaerobic loop,

0.76 � 0.26 and 1.58 � 0.23 mg N L�1, respectively. This

difference was statistically significant (ANOVA, p < 0.03). POM

C/N ratios were significantly (ANOVA, p < 0.002) higher in the

aerobic loop than in the anaerobic loop, 6� 0.24 and 4.9� 0.21,

respectively.

3.1.3. Digestion basin profilesDO in the surface water of the DB decreased from the DBin to

the DBout. A decrease in oxygen was also observed with depth

(Fig. 3; a representative profile from the DB center measured

on 6/11/2006). It was found that oxygen was totally consumed

within the upper 5 cm of the sludge layer. These findings were

Table 1 e The POM isotopic values (&) and the C and N concentrations (mg/L) measured in the water at the mariculturesampling stations, abbreviation as in Fig. 1. The results are expressed as average ± SD.

Parameter FB TFB TFout TFin DBinT DBinB DBoutT DBoutB FBR

d13CPOM �23.03 �22.96 �23.18 �23.03 �23.35 �23.16 �23.10 �23.59 �23.11

SD 1.52 1.16 1.03 0.83 1.04 0.92 2.01 2.09 2.01

d15NPOM 7.63 9.03 8.58 9.20 7.42 7.47 7.51 7.23 7.57

SD 2.23 2.32 2.50 2.49 3.64 3.13 4.33 3.52 3.31

CPOM 2.31 2.71 2.72 2.32 3.90 4.24 3.68 4.58 3.87

SD 1.53 2.39 2.13 1.85 1.97 2.63 2.26 3.35 3.17

NPOM 0.52 0.60 0.52 0.51 0.95 0.79 0.87 1.14 0.93

SD 0.34 0.52 0.45 0.38 0.25 0.55 0.35 0.59 0.57

C/NPOM 5.97 6.50 5.84 5.77 4.88 4.94 4.82 5.00 4.93

SD 1.69 2.35 1.21 1.02 0.38 0.34 0.17 0.39 0.24

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 2 3 7 5e2 3 8 22380

further validated by redox potential measurements showing

a similar profile (Fig. 3A). A representative nutrient profile of

the center of the DB (Fig. 3B) showed a nitrate decrease from

the surface to the bottom with depletion at about 15 cm into

the sludge. TAN and sulfide profiles showed an opposite trend

as compared to nitrate.

3.1.4. d13C and d15N values in sludged13Csludge and d15Nsludge values were �22.5 � 0.9& and

9.2 � 1.3&, respectively. d13Csludge and d15Nsludge did not show

any significant difference between the different areas within

the DB and the organic matter derived from the wall of the

TFB. The average values of the d13Csludge were very similar to

that of the fish feed�22.2� 3& (ANOVA, p< 0.67), while those

of the d15Nsludge were 15N enriched compared to fish feed

6.2 � 0.5& (ANOVA, p < 0.0001). The C/N ratio of the sludge

was slightly lower than that of fish feed 6 � 0.31 and

6.64 � 0.26, respectively (ANOVA, p < 0.01).

4. Discussion

Significant differences in the inorganic carbonate systemwere

found between the aerobic and anaerobic loops. Higher DIC

and alkalinity, and 13C depleted d13CDIC values were measured

in the anaerobic loop than in the aerobic loop (Fig. 2).

Considering the main processes taking place in the treatment

systems,we suggest that in the anaerobic loop 13C depletion in

the DIC is caused by a CO2 release through respiratory

processes and by an alkalinity increase through denitrification

(van Rijn et al., 2006). In the aerobic loop, alkalinity is

consumed due to nitrification (Chen et al., 2006; Eding et al.,

2006) by chemolithoautotrophic microorganisms that assim-

ilate 13C depleted CO2 from the water (Foesel et al., 2008;

Sakata et al., 2008). This CO2 assimilation and, in addition,

the intensive CO2 degassing taking place as a result of the low

water retention time and the specific configuration of the

trickling filters (Eding et al., 2006), may provide an explanation

for the 13C enrichment in the aerobic treatment compartment.

It seems, therefore, that the difference in alkalinity, CO2

uptake/release and the consequent difference in buffering

capacity as well as the differences in filter configuration and

retention time are responsible for the difference in DIC values

between the two loops. Lower DIC values were measured in

the TF components than in the fish basin. Within the aerobic

loop, lowest DIC values were measured in the TFout. This is

consistent with the chemolithoautotrophic utilization of CO2

and the consumption of alkalinity by the nitrification process

within the TF. Based on themethodology used in this study as

well as results from previous studies, which demonstrated

oxidation of TAN to nitrate within the filter (van Rijn and

Rivera, 1990; Gelfand et al., 2003), it might be concluded that

autotrophic nitrification is a major process within this filter.

Organic matter degradation in the DB is rapid. It was

estimated that during one growth season (around one year)

about 90% of the total organic matter added to the system

and not utilized by fish, is digested (Fine, unpublished data;

van Rijn et al., 1995; Gelfand et al., 2003; Neori et al., 2007).

The d13CDIC in the anaerobic loop is 13C depleted compared to

the aerobic loop by 2.5e3&. This finding is consistent with

the relative 13C depleted organic matter respired and miner-

alized in the DB. A bigger difference would be expected but, as

previously noted, the difference in DIC between the aerobic

and anaerobic loops is controlled by alkalinity and the water

retention time in each of the loops. Both factors directly

affect the efficiency of CO2 degassing, which is a dynamic

process. Based on thermodynamic considerations, a signifi-

cant kinetic discrimination against 13C is expected due to this

process. The relative small difference between the loops may

be explained by 13C depleted CO2 gas escaping from the DB

and degassing during the water return to the aerobic loop due

to the lower alkalinity and low water retention time there. It

should be emphasized that, despite its static plug-flow mode

of operation, large quantities of CO2 are released into the

atmosphere in the DB since CO2 generation in this reactor is

high. CO2 generation in the DB is high as feces and uneaten

feed are all diverted to this basin. How much CO2 is released

by digestion of the feces and uneaten feed can roughly be

estimated by assuming that fish utilize around 50% of the

carbon supplied with the feed (Neori et al., 2007). In this

particular system, feed loads were as high as 4 kg per day

thus, without accounting for uneaten feed, at least 1 kg of

carbon was daily added to the DB. In the DB around 90% of

the carbon is digested (Neori et al., 2007) which means that

0.9 kg carbon in the form of CO2 is produced daily. This daily

added amount of carbon to the DB is equal to about 3e4 times

the average amount of DIC in the DB, which can be calculated

from DIC concentrations (Fig. 2) and the water volume of the

DB. If no CO2 gas is released from the DB, it would be added to

the DIC in the water. In such a case, the only exchange of DIC

in the DB would be by water exchange. Because of the long

water retention time in the DB (4.5 h), the d13CDIC would

equilibrate to values similar to that of the organic material

added to the DB i.e. w�22&, as opposed to measured values

of w�10&. The carbonate system is probably in equilibrium

between the DIC concentration determined by its alkalinity

and the pCO2 in the atmosphere due to a relative long

retention time of the DB. Based on these findings it seems

likely, therefore, that the difference in d13CDIC between the

aerobic and the anaerobic loops is controlled by the alkalinity

differences.

The fish in the mariculture are fed with pellets (48%

protein, 20% fat) as the single external source of organic

matter and feed, therefore, it is the source of most of the new

organic matter in the system. The only other new source of

carbon in the system is CO2 fixed by autotrophic bacteria,

which in this particular system are mainly represented by

nitrifying bacteria in the TF. Most of the organic matter

produced in the TF is removed by the foam fractionator and is

disposed in the DB, but quantitatively this source is highly

insignificant. Feed pellets d13C and d15N values were

�22.2 � 3& and 6.2 � 0.5&, respectively, while in the fish

tissue these values were �20.4 � 1.2& and 10.8 � 0.6&,

respectively, thus showing an increase in the isotopic values

of about 2& and 4& in the d13C and d15N, respectively. These

findings are consistent with previous studies, which point to

an increase in d13C and d15N with each trophic level (DeNiro

and Epstein, 1978; Fry and Sherr, 1984; Minagawa and Wada,

1984) and confirming that the fish in this system use feed

pellets as their sole source of nutrition.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 2 3 7 5e2 3 8 2 2381

The input of organicmatter from the fish basin to the DB as

POM consists of undigested feed pellets and fish feces. This

organic matter settles in the DB and serves as substrate for

microbial respiratory processes mainly using nitrate and

sulfate as electron acceptors as can be seen from the chemical

and redox potential profiles in the DB (Fig. 3). The DB average

d13Csludge and d15Nsludge values were �22.5 � 0.9& and

9.2 � 1.3&, respectively and the d13CPOM and d15NPOM were

�23.3 � 1.4&, 7.4 � 3.3&, respectively. The carbon isotopes

values in the DB sludge and POM were not significantly

different from that of the pellets, while the nitrogen isotopes

were 15N enriched compared to the pellets. The enrichment in

the nitrogen isotopes may be a result of microbial decompo-

sition (Fellerhoff et al., 2003), probably releasing 15N depleted

ammonia. This possibility is further substantiated by the high

TAN (Fig. 3B) in the bottom layers of the sludge column in

agreement with previous work on a smaller scale system

(Gelfand et al., 2003). It was shownby Cytryn et al. (2003), using

DNA sequence analysis from the DB sludge, that a number of

dominant microorganisms were affiliated with fermentative

bacteria, Fusibacteria, Dethiosulfovibrio and members of the

Bacteroidetes phylum. These fermentative bacteria are involved

in the degradation of macromolecular compounds whereby

secondary metabolites such as volatile fatty acids (VFA) are

liberated (van Rijn et al., 1995). Under these conditions, liber-

ated VFAwere shown to undergo a rapid oxidation by bacterial

respiration with mainly sulfate and nitrate as electron

acceptors (Aboutboul et al., 1995; van Rijn et al., 1996, 1995).

5. Conclusions

In this study, an approach, based on integrating stable

isotopes with additional chemical analysis, was used to trace

carbon in a zero-discharge mariculture system. It was shown

that alkalinity values provide a clear indication for the main

microbiological processes taking place in each of the system

components. The carbon (DIC and POM) and nitrogen (POM)

values show a consistent difference between the aerobic and

anaerobic loops caused by a combination of differences in

microbial processes and water retention time in these loops.

Further studies are required to determine how d13C profiles,

POM formation and different respiratory pathways affect the

isotopic values in the system. Once such links are established,

the technique of stable isotope tracing has the potential to

diagnose changes in systems, such as examined in this study,

by means of a few relatively simple measurements.

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